Proceedings of the Thirty-Fourth U.S.-Japan Aquaculture Panel Symposium

Aquaculture and Stock Enhancement of Finﬁsh Proceedings of the Thirty-fourth U.S.-Japan Aquaculture Panel Symposium

San Diego, California November 7–9, 2005

Robert Stickney, Robert Iwamoto, and Michael Rust, editors

U.S. DEPARTMENT OF COMMERCE National Oceanic and Atmospheric Administration National Marine Fisheries Service

NOAA Technical Memorandum NMFS-F/SPO-85

Aquaculture and Stock Enhancement of Finﬁsh Proceedings of the Thirty-fourth U.S.-Japan Aquaculture Panel Symposium

San Diego, California November 7–9, 2005

Robert Stickney, Robert Iwamoto, and Michael Rust, editors

NOAA Technical Memorandum NMFS-F/SPO-85 October 2007

U.S. Department of Commerce Carlos M. Gutierrez, Secretary

National Oceanic and Atmospheric Administration Vice Admiral Conrad C. Lautenbacher, Jr., USN (Ret.) Under Secretary for Oceans and Atmosphere

National Marine Fisheries Service William T. Hogarth, Assistant Administrator for Fisheries Suggested citation: Stickney, R., R. Iwamoto, and M. Rust (editors). 2007. Aquaculture and Stock Enhancement of Finﬁsh: Proceedings of the Thirty-fourth U.S.-Japan Aquaculture Panel Symposium, San Diego, California, November 7–9, 2005. U.S. Dept. Commerce, NOAA Tech. Memo. NMFS-F/SPO-85, 76 p.

A copy of this report may be obtained from: Northwest Fisheries Science Center 2725 Montlake Boulevard East Seattle, Washington 98112

Or online at: http://spo.nmfs.noaa.gov/tm/

Reference throughout this document to trade names does not imply endorsement by the National Marine Fisheries Service, NOAA. ii Table of Introduction Contents Species Reports Seriola Current Situation of Technical Developments in Seed 1 Production of Yellowtail (Seriola quinqueradiata) in Japan Keiichi Mushiake, Hideki Yamazaki, and Hiroshi Fujimoto

Sciaenids Culture of Spotted Seatrout (Cynoscion nebulosus) in a Closed, 5 Recirculating System Reginald B. Blaylock, Angelos Apeitos, Jason T. Lemus, and Jeffrey M. Lotz

Disease Surveillance in Wild and Cultured Stocks of White 11 Seabass (Atractoscion nobilis) Mark S. Okihiro

Sebastes Juvenile Mass Production of Black Rockﬁsh (Sebastes 21 schlegeli) in FRA Miyako Station: Present Status and Problems Masahiro Nakagawa, Junichi Adachi, Ken Shimizu, and Satoshi Shiozawa

Paralichthys Construction of a BAC Library from XY Japanese Flounder 27 Using Frozen Sperm Genomic DNA Hiroyuki Okamoto, Hiroyuki Nagoya, Eiichi Yamamoto, Takashi Sakamoto, Kanako Fuji, Nobuaki Okamoto, Kazuo Araki, and Ichiro Nakayama

Scombrids Status of Paciﬁc Blueﬁn Tuna (Thunnus orientalis) Seed 31 Production in Amami Station of NCSE FRA Hideki Nikaido, Takayuki Takebe, Kentaro Ide, Hitoshi Imaizumi, Nobuhiro Tezuka, and Syukei Masuma

Genetics and Reproduction Visualization Tools to Probe Early Stage Fish Abnormalities 37 Susumu Uji and Tadahide Kurokawa

Special Topics Airlift-PolyGeyser Combination Facilitates Decentralized 43 Water Treatment in Recirculating Marine Hatchery Systems Ronald F. Malone and Sripavani Gudipati

Preparation of Marine Silage and its Potential for Industrial 51 Use Motoharu Uchida

iii Special Topics (continued) Techniques for Live Capture of Deepwater Fishes with Special 57 Emphasis on the Design and Application of a Low-cost Hyperbaric Chamber Jeffrey E. Smiley and Mark A. Drawbridge

A Preliminary Report on the Use of Porphyra Protoplast as a 63 Food Substitute for Culturing Aquatic Animals TakaoYoshimatsu, Alok Kalla, Toshiyoshi Araki, Dong-Ming Zhang, and Shuichi Sakamoto

Aquaculture and Stock Enhancement Technologies Based on 69 Recently Discovered Calcium-sensing Receptors in Finﬁsh Steve Jury, H. William Harris, Timothy Linley, David Russell, Marlies Betka, Susan Anderson, Shinji Harakawa, and Akikuni Hara

Biological Studies on the Digestive Mechanism in Abalone 75 Kentaro Niwa, Hideaki Aono, and Tomoo Sawabe

iv Introduction The U.S.-Japan Cooperative Program in Natural Resources (UJNR) was established on January 29, 1964. Since its creation, the UJNR has evolved to become one of the oldest and most effective cooperative agreements between Japan and the United States. In 1969, the UJNR Aquaculture Panel was created as a vehicle for scientists of both countries to meet and discuss aquaculture research accomplishments, needs, and priorities, as well as provide opportunities for cooperative research and scientiﬁc exchange.

Under the auspices of the UJNR Aquaculture Panel, U.S.-Japan scientists have met annually since 1971 without interruption. The venue for these meetings has alternated between the two countries. In November 2005 the meeting (comprised of a business meeting, scientific symposium, and several field trips) was held at the Hubbs-SeaWorld Research Institute, San Diego, California, and nearby environs. The scientific symposium was coordinated by Mark Drawbridge of Hubbs Sea-World and Dr. Kenneth Leber of Mote Marine Laboratory, Sarasota, Florida. By prior agreement of the U.S.-Japan panels, contributors to the proceedings were requested to submit manuscripts in the form of extended abstracts.

Yasuji Sakai, Japan Robert Iwamoto, United States

v vi Current Situation of Technical Developments in Seed Production of Yellowtail (Seriola quinqueradiata) in Japan

Keiichi Mushiake Headquarters, Fisheries Research Agency 15th Floor Queen’s Tower B 2-3-3 Minatomirai, Nishi-ku, Yokohama Kanagawa, Japan, 220-6115 [email protected]

Hideki Yamazaki Yashima Station of National Center for Stock Enhancement FRA, Takamatsu Kagawa, Japan, 761-0111

Hiroshi Fujimoto Obama Station of NCSE FRA, Obama Fukui, Japan, 917-0117

Keywords: Yellowtail, Seriola quinqueradiata, seed production, broodstock management

Abstract The National Center for Stock Enhancement (NCSE, formerly Japan Sea-Farming Association), of the Fisheries Research Agency, introduced the stock enhancement program for yellowtail (Seriola quinqueradiata) in 1977. Technical developments in induced spawning as well as larval and juvenile rearing techniques have increased the production of this species to 1 million juveniles per year at NCSE. This project faced three major drawbacks: high mortality of larvae, cannibalism, and the smaller size of released juveniles in comparison with their wild counterparts. The high mortality of larvae was overcome by utilizing strong aeration during the early larval stages, while cannibalism was controlled by grading juveniles by size selection. The two-month delay in the spawning season of reared broodstock (the usual spawning season is late April to early May), which caused the smaller size of released juveniles, was solved by developments in advanced spawning techniques. Photoperiod and water temperature manipulations were used to produce eggs in February, thus producing yellowtail juveniles that can be released into the wild at a size similar to that of the wild stock.

Introduction Yellowtail (Seriola quinqueradiata), which is a Research Agency (FRA), started to develop the large migratory species that moves north and south in techniques for broodstock management of yellowtail waters adjacent to the Japanese archipelago, is an in 1977. In 1978 Yashima and Kamiura Stations of important species of the fishery stock in western NCSE started seed production of the species, Japan and the coastal areas of the Sea of Japan. In followed by Goto Station of NCSE in 1981. 2002, the annual catch was 50,000 tons and the Yellowtail is a unique species distributed only aquaculture industry produced 140,000 tons. near the coast of Japan. The major spawning ground Because there is apprehension that the yellowtail is the south-central area of the East China Sea where stock may be reduced by capture of wild juveniles the spawning season starts around February. In (called mojako) for aquaculture, yellowtail juveniles Shikoku and the northern part of Kyushu, it starts were chosen as a target species for sea ranching in around April or May, so the season becomes later as 1977. Komame and Goto Stations of the National one goes further north (Hamada and Mushiake, Center for Stock Enhancement (NCSE, formerly 2006). Japan Sea-Farming Association), of the Fisheries

Mushiake 1 Brief history of seed production in yellowtail current that prevented the larvae from accumulating Collection of eggs and culture of larvae and at the bottom of the tanks. The method markedly juveniles were started in order to obtain basic increased the survival rate up to 10 days after information in 1960. A marked decrease of catch hatching. Compared with the previous method, the quantity in 1970s led NCSE to begin the technical survival rate at the early stage was increased from development for broodstock management in 1977 21.7% to 58.1% in 1995 and from 14.6% to 57.4% in and seed production in 1978. 1997. In NCSE, the brief history of the technical The other problem was cannibalism. The best developments for yellowtail culture was as follows way to prevent cannibalism is to grade the juveniles (Shiozaw and Yamasaki 2006): (Shiozawa and Yamasaki 2006). When yellowtail 1. 1977–1989. NCSE was engaged in seed reach 10 mm total length (TL), they go up to the production in large quantities, thereby surface at night, so grading is carried out during a enabling the production of one million single night when the fish reach around 15 mm TL, juveniles in just one station by 1986. as that is the size when cannibalism is known to start. 2. 1990–1995. The technical developments The floating, sleeping juveniles are siphoned out of focused on the quality of seed production, the rearing tank and transferred to a net pen of 3 mm rather than quantity, through the introduction mesh size in another tank. Only individuals that of formulated diets and developments in the couldn’t pass through the mesh remain within the net improvement of survival rates during the thereby separating the fish into two size groups. early stages. Figure 1 shows the total length distribution in the 3. 1996–2005. Since 1996 advanced egg large and small sized fish that were selected by this collection and early seed production method. The fish were clearly divided into two techniques have been implemented. groups at around 18 mm and the positive effects of In the present paper, some technical developments in selection are shown by the survival rate. This size seed production since 1990 are presented, as well as selection at night has clearly doubled the survival the details of both improvement techniques for rate, when compared to the nongraded fish. That is, survival in seed production and advanced spawning according to size selection at 15 mm TL, the survival of broodstock. rate of 15–30 mm TL was improved from 33.8% to 78.9% in 1994 and from 24.4% to 52.1% in 1995. At Improvement techniques for survival in seed present, survival rates of around 15% at 30 mm in TL production of yellowtail are obtained. In exceptional cases, the survival rate The drawback of seed production for yellowtail can even reach up to 30%. was the high incidence of larval mortality that occurred during the early larval stages. Moreover, cannibalism was observed from when they reached a total length of about 15 mm until they grew to juvenile size. Those were the two major causes of mortality, and some measures have been taken to counteract the problems. One approach was aeration which led to a high survival rate during the early larval stages. In the past, the sinking of hatched larvae, seen before and after the mouth opening stage, was a major problem leading to a high mortality (Shiozawa and Yamasaki. 2006). With the conventional method, slight aeration was used during the early stages of rearing. The water in the tank was slightly up-welled vertically, Figure 1. Size frequency (TL) of yellowtail larvae graded though only locally in the tanks, leading to high with a 3 mm mesh net at night when the fish averaged 15 mm mortalities due to the sinking of larvae. Since 1995, TL. the problem has been eliminated by employing stronger aeration using air-block pipes set in each corner at the bottom of the tanks. This method enabled the tank water to have a stronger upwelling

2 Mushiake Advanced spawning of yellowtail broodstock hatchery-reared juveniles that were produced Broodstock of wild origin were reared in net by advanced spawning showed growth rates cages and used for egg collection. Egg collection to their wild counterparts. These developed was mainly carried out from late April to early May techniques were effective for the promotion until 1995 (Mushiake 1994). The collection was of stock enhancement. carried out after the broodstock were maintained at 3. At present, yellowtail aquaculture depends 19°C. When the oocyte diameter reached over 700 on captured wild juveniles. However, there micrometers, the broodstock were injected with 600 is a concern that a decrease in wild juveniles IU/kg body weight with human chorionic may have some adverse effect on the gonadotropin (HCG). The procedure enables egg fundamental management of yellowtail collection 48 hours after the hormone injection. The aquaculture. According to our studies, there hormone should be injected when broodstock was no difference between yellowtail maturity is in the last phase of yolk deposition. produced in advanced spawning and those About one million eggs were obtained. However, the hatched in the wild. The next challenge will size of hatchery-reared juveniles was much smaller be to utilize the hatchery seed of yellowtail than that of their wild counterparts that were caught for aquaculture purposes. during the same period because of a two month delay in the spawning season of reared broodstock. Therefore in 1996, in addition to the previous rearing methods for captive broodstock, a rearing environment that had short and long photoperiods and manipulated water temperatures was provided in order to induce earlier maturation. These manipulations were followed by HCG injection and spawning was successfully induced between February and March (Mushiake et al. 1994, 1998). Now eggs are available for seed production in the hatchery at the same time as the ones that are spawned naturally in the wild. As shown in Figure 2, the growth of the hatchery- reared juveniles originated from the advanced Figure 2. Growth of hatchery-reared juvenile yellowtail originating from advanced spawning in February (closed spawning on February, as represented by closed circles) compared with wild spawned yellowtail (open circles). circles, clearly showed that the juveniles had a similar size and almost the same growth as their wild counterparts, as indicated by open circles. In fact, the hatchery-reared juveniles were released with a brand mark into the Seto Inland Sea and collected at a fish market in December 1999. The body weight of the wild and the released fish was 1.2 kg and both types traded for the same price in the fish market. The seed production at present is capable of producing fish of a similar size to the wild stock by utilizing eggs obtained from advanced spawning in February (Fujimoto et al. 2006). Thus far, the following technical developments were shown in the present study. 1. The methods of egg collection and seed production techniques were the optimal ones

and solve the various problems faced in the past, so the techniques have been already established for yellowtail. 2. With regard to the advanced egg collection method during February and March, the

Mushiake 3 References Fujimoto, H., H. Yamasaki, and A. Suda. 2006. Release Mushiake, K., K. Kawano, W. Sakamoto, and I. for stock enhancement. In M. Matsuyama, Y. Hiyama, K. Hasegawa. 1994. Effect of extended daylength on ovarian Mushiake, and H. Yoshiaki (eds.), Biology, stock maturation and HCG-induced spawning in yellowtail fed enhancement and aquaculture of buri [Japanese yellowtail moist pellets. Fish. Sci. 60:647–651. (Seriola quinqueradiata)]. Mushiake, K., K. Kawano, K. Kobayashi, and T. Hamada, K., and K. Mushiake. 2006. Advanced Yamasaki. 1998. Advanced spawning in yellowtail, spawning of yellowtail Seriola quinqueradiata as early as Seriola quinqueradiata, by manipulations of the December by manipulations of both photoperiod and water photoperiod and water temperature. Fish. Sci. 64:727–731. temperature. Nippon Suisan Gakkaishi, 72(2):186-192. Shiozawa, S., and H. Yamasaki. 2006. Seed production of Mushiake K. 1994. Studies on technical development of juveniles. In M. Matsuyama, Y. Hiyama, K. Mushiake, broodstock management in striped jack and yellowtail. and H. Yoshiaki (eds.), Biology, stock enhancement and Doctoral thesis. Hiroshima University, Higashi-hiroshima. aquaculture of buri [Japanese yellowtail (Seriola quinqueradiata)].

4 Mushiake Culture of Spotted, Seatrout (Cynoscion nebulosus) in a Closed, Recirculating System

Reginald B. Blaylock, Angelos Apeitos, Jason T. Lemus, and Jeffrey M. Lotz Gulf Coast Research Laboratory The University of Southern Mississippi P.O. Box 7000 Ocean Springs, Mississippi 39566-7000 [email protected]

Keywords: Spotted seatrout, intensive culture, recirculation, Cynoscion nebulosus, larviculture

Abstract The spotted seatrout (Cynoscion nebulosus) is the most popular sport fish among anglers in the estuarine and nearshore waters of Mississippi. In partnership with the Mississippi Department of Marine Resources, we have begun to evaluate the feasibility of using cultured spotted seatrout to supplement the natural stocks in Mississippi waters. Presently, culture of seatrout consists of captive spawning of broodstock and rearing of larvae in earthen ponds containing estuarine water and mixed wild zooplankton. We have designed a recirculating system in which to rear spotted seatrout larvae and juveniles intensively. As a test of the intensive culture system, approximately 80,000 spotted seatrout (2 day posthatch [PH]) larvae were obtained from Sea Center Texas in Lake Jackson. Larvae were stocked in eight, 1,000 L tanks at a density of 10 larvae/liter. Feed consisted of ss-rotifers (range 70-110 µm), Artemia nauplii, and dry food. Mean survival from day 2 PH through day 25 PH was 25% and the larvae had a mean total length of 16.64 mm ±0.79 mm and a mean weight of 0.04 g ±0.01 g. Cannibalism commenced at day 7 PH. At day 24 PH the juveniles were transferred to a nursery system. At day 38 PH the fish were transferred to a larger 15×3.5×1 m, 55,000 L raceway. Survival during the nursery phase averaged 88.84% ±5.80%. Larvae averaged 45.01 mm ±1.93 mm and 0.79 g ±0.10 g.

Introduction Worldwide, marine fish populations are managed stocks, and an accumulation of data from declining. Current capture fisheries, while relatively studies in Japan (Tsukamoto et al. 1989, Tsukamoto stable over the last 15 years, may be unsustainable 1993), Norway (Svasand et al. 1990), and Hawaii past the year 2040 (New 1997). Of the fish (Leber 1995) inspired Blankenship and Leber (1995) populations that have been assessed, more than 30% to revive the idea that successful marine stock are currently overfished (Pew Oceans Commission enhancement is possible through carefully planned 2003). In response to the growing problem, the research that incorporates principles of fish culture, Magnuson-Stevens and Sustainable Fisheries acts ecology, health, genetics, and assessment. required that plans for management and restoration of The spotted seatrout (Cynoscion nebulosus) is an overfished species be put in place. Typically, the estuarine and nearshore species ranging from New plans include catch/size limitations or habitat York to Texas (Robins and Ray 1986). In the restoration, but additional techniques to assist southeastern U.S. and in the Gulf of Mexico, the management agencies are needed. spotted seatrout is widely esteemed among Stock enhancement—the release of cultured fish recreational anglers. In Mississippi waters, the into the wild—actually served as the technique of spotted seatrout is the premier sport fish. However, choice in marine fisheries management throughout increasing human population places pressure on most of the nineteenth and twentieth centuries. marine fisheries resources through habitat destruction However, the lack of evidence of effectiveness and demand for food and recreation. The popularity combined with concerns over maladaptive behaviors, of seatrout, combined with its dependence on artificial genetic selection, and disease problems in threatened inshore habitats, make it potentially cultured fish created skepticism about the desirability vulnerable to depletion. It is from this perspective of the approach. The advent of new hatchery and that the Gulf Coast Research Laboratory in tagging technologies, the continued decline of cooperation with the Mississippi Department of Blaylock 5 Marine Resources and recreational angler and maintaining broodstock; 2) Spawning—the Organizations founded SPEC, the Seatrout development of methods for conditioning healthy Population Enhancement Cooperative, a program to broodstock for natural spawning in captivity; 3) determine whether stock enhancement is a feasible culture—the development of methods to maximize option for management of spotted seatrout in production of fish that can be tagged for release in Mississippi. intensive, indoor recirculating systems; 4) tag and In this paper, we present the framework of the release—the development of technology to efficiently program we developed for the intensive rearing of and reliably tag and release juveniles; and 5) spotted seatrout for stock enhancement in Mississippi postrelease assessment—the development of methods waters. to monitor juveniles after release and analyze the information to assess the effectiveness of the Seatrout culture program. The culture of seatrout is not new. Wild seatrout juveniles were stocked into brackish ponds with Broodstock development. Broodstock development mixed results as early as the 1940s (Colura et al. is patterned directly after the Texas Parks and 1992). Colura (1974) first described induced Wildlife program (David Abrego, personal spawning of spotted seatrout. Arnold et al. (1976) communication). Wild animals are caught locally on and Taniguchi (1981) described methods for hook-and-line using barbless hooks. Seatrout are spawning and rearing spotted seatrout in the delicate animals and must be handled with great care. laboratory. Colura et al. (1976) and Tucker (1988) Soft catch-and-release nets or standard nets lined described experimental methods of fingerling with plastic are required for landing the fish. Fish are production for spotted seatrout. Although Arnold’s transferred from the hook into holding pens that serve original production system included early larval as a reservoir to contain all fish caught during a rearing in which he achieved as high as 30% survival collecting event. Upon completion of the collecting through 30 days, his system evolved into the event, fish are transferred using the plastic-lined nets extensive pond culture approach described in Colura into hauling tanks for transport to the lab. Stress Coat et al. (1992) whereby prefeeding larvae are and supplemental oxygen are added to the hauling introduced into brackish water ponds containing wild, tank. It is important to note that at no point during mixed zooplankton. Part of the reason for this process are the fish removed from the water. The transitioning to pond rearing was inconsistent and population structure of seatrout in Mississippi is not low yields in laboratory experiments. Colura’s yet known, so until we evaluate the population extensive system produced an average survival of structure, we assume animals from the Pascagoula 28.7% over 20–30 days of rearing (Colura et al. River, the Biloxi Bay area, and Bay St. Louis are 1992). The Texas Department of Parks and Wildlife distinct populations. currently uses a variation of this method to produce Upon arrival at the laboratory, fish are placed in approximately 3 million 30-day-old larvae annually. a staging area that allows acclimatization and Since 1993 Texas has released approximately 35 recovery from the stress of capture and transport. million 30-day-old juvenile seatrout (David Abrego, After a brief acclimatization period, fish enter the personal communication). quarantine area. Because our goal is to maintain fish In coastal Mississippi, which is estuarine, pond in optimal condition for long-term holding and culture is not feasible for a variety of reasons, because all wild fish have parasites, we initially including the highly variable salinities, the lack of quarantine the fish to eliminate diseased fish and available space, and the shallow water table. Thus parasites that are likely to cause problems in captivity our program has focused on intensive tank culture. (i.e., parasites with direct life cycles). We practice As a result, the process is more complex and labor standard animal care facility sanitation practices such intensive than pond rearing; however, preliminary as access control, hand/foot/equipment disinfection, results suggest that the process produces a result at and barrier protection after the quarantine period. least as good as extensive culture. Quarantine. Quarantine consists of three stages. Mississippi program First, fish enter pretreatment to remove ectoparasites. Our program focuses on five primary objectives: Pretreatment consists of two steps, a 5 minute 1) broodstock development—the development of freshwater dip that detaches many of the technology for capturing, transporting, quarantining ectoparasites, particularly Amyloodinium ocellatum,

6 Blaylock followed by a 24 hour treatment with Praziquantel and upon hatching (≈48 hours) the larvae are placed (2.5–5.0 mg/l) to rid the fish of dangerous at a density of 10/L in 1,000 L larval rearing tanks monogeneans. Following pretreatment, fish enter previously stocked with 50,000 cells/ml of live primary quarantine in a 12,000 L raceway for a Tahitian strain Isochrysis galbana. Beginning on day minimum of 10 days, during which they are observed t2 posthatch (PH), enriched ss-rotifers at 10/ml and for health problems (and treated if necessary) and cultured copepod nauplii at 0.5-1.0/ml are added. On transitioned to frozen food. Seatrout are slow to day 7 PH enriched brine shrimp nauplii are offered at begin feeding in captivity, so after a few days of 1/ml. Copepods are discontinued on day 8 PH and isolation we slowly introduce live, laboratory-reared rotifers are discontinued on day 9 PH. Brine shrimp shrimp. Once the fish begin to eat the live shrimp, are increased to 2/ml on day 10 and to 4/ml on day 13 frozen shrimp are added. Eventually, frozen squid PH. Sprinkles of dry, size-00 mash are offered are added. Ten to 14 days after entry into primary beginning on day 15 PH and the amount is slowly quarantine, the fish are treated with formalin (175 increased over time. Brine shrimp are decreased to mg/L for 60–90 min) to remove any residual 2/ml on day 18 PH. Dry, size-0 crumble concurrent ectoparasites. After formalin treatment, the fish enter with 00-mash begins to be offered on day 20 PH and the third stage—secondary quarantine—where they size-1 crumble is added to the mixture on day 23 PH. are observed and retreated as necessary for an Harvest to the nursery occurs on days 24 and 25 PH. additional 20 days. Of the larvae stocked into the system, we achieved approximately 25% survival through day 24 PH. Maturation. After quarantine, fish enter the maturation facility. During the move, the fish are Nursery phase. Trout are typically released into anesthetized, weighed, measured, sexed, tagged ponds after the initial culture period; thus, protocols according to sex, and fin clipped for DNA analysis. for seatrout nursery operations are largely Fish are stocked into 12,000 L circular tanks fitted experimental, particularly for large-scale production. with egg collectors in a temperature and photoperiod Our system is based on that used in our red snapper controlled environment. Water quality is maintained culture operation (Ogle and Lotz 2005) and consists using a suspended trickle bioball biofilter, sand filter, of fourteen, 1,000 L tanks equipped with adjustable coral filter, protein skimmer, and ozonation unit. spray bars. Cannibalism began on day 7 of the study. Each tank is stocked with 20 fish, ideally in a 1:1 sex By day 24, cannibalism was a significant source of ratio. The fish are fed a combination of shrimp, mortality. We did two things in the nursery to try and squid, and cigar minnows at 3% of body weight three minimize the problem. First, we used the adjustable times per week using the Texas Parks and Wildlife spray bars and pumps to create currents sufficient to protocol (David Abrego, personal communication). keep the juveniles active. Second, we manipulated The ratio of shrimp, squid, and cigar minnows varies the density. Not all species respond to changes in depending on season. During spawning, animals are density in the same manner (e.g., Kestemont et al. fed shrimp (≈36%), squid (≈45%) and cigar minnows 2003) and the response of seatrout was unknown. (≈18%). Postspawning animals are fed at the same Thus we initially stocked at three different densities rate a diet consisting of approximately 16% shrimp, (four tanks at 0.8 fish/L, four at 1/L, four at 1.5/L, 42% squid, and 42% cigar minnows. The and two at 3.5/L). Initially, size-1 crumble was temperature and photoperiod cycle mimics the slowly sprinkled into the tanks to acclimatize the fish, natural cycle, but is condensed to 150 days based on but food level was increased to 3% body weight per the Texas Parks and Wildlife protocol to provide day based on the average weight of a sample of quick turnaround or off-season spawning. juveniles going into the nursery. Originally, the nursery phase was intended to last for at least 14 Larviculture. Our larviculture system is a days, however concerns over our ability to maintain modification of the combined experiences reported in water quality in the high density tanks led to Arnold et al. (1976), Taniguchi (1981) and Tucker harvesting at day 10 of the study (~35 days PH). We (1988) as well as the literature on the culture of other achieved an average of 88.8% survival (range of marine fishes including the red snapper (Ogle and 79.8–95.7%), and although the average survival was Lotz 2005). Seatrout spawn at night. The eggs are lower in the high density tanks (82.4%), the buoyant and are carried with the current out of the variability was such that we can not summarily tank into an egg collector where they are retained. exclude a high density nursery as an option for future From the egg collector they are placed in a conical batches of juveniles. Although fish stocked at the incubator and agitated with air. Viable eggs float, highest density were smaller than those stocked at Blaylock 7 other densities, major size discrepancies within tanks References were not observed. Arnold, C. R., J. L. Lasswell, W. H. Bailey, T. D. Williams and W. A. Fable, Jr. 1976. Methods and techniques for Growout. Growout for this batch was accomplished spawning and rearing of spotted seatrout in the laboratory. in a 15×3.5×1 m rectangular raceway (approximately Proc. Annu. Conf. Southeast Assoc. Fish Wildl. Agencies 50,000 L capacity), equipped with suspended trickle 30:166–178. bioblock biological filters, sand filters and a protein Blankenship, H. L., and K. M. Leber. 1995. A responsible skimmer. Fish were fed progressively larger pellets approach to marine stock enhancement. Am. Fish. Soc. at approximately 3% body weight based on periodic Symp. 15:167–175. samples. All fish died at approximately day 60 PH during hurricane Katrina. The juveniles were Colura, R. L. 1974. Induced spawning of the spotted approximately 80–100 mm, and we estimated seatrout, Cynoscion nebulosus (Cuvier). Proc. World approximately 75% survival to day 60. There was Maricul. Soc. 5:319–330. more size variability than in the nursery, perhaps due to unequal access to food created by poor circulation Colura, R. L., B. T. Hysmith, and R. E. Stevens. 1976. in the raceway. Fingerling production of striped bass (Morone saxatilis), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus) in saltwater ponds. Proc. World Tagging and release. At the time of the hurricane, Maricul. Soc. 7:79–92. plans for experimental tagging with coded-wire and elastomer tags to assess handling and insertion Colura, R. L., T. L. King, J. D. Gray and B. W. procedures, tag placement, and tag retention were Bumguardner. 1992. Analyses of six years of spotted being developed. Acoustic tags will be explored in seatrout (Cynoscion nebulosus) pond culture trials. the future. Growth rates and feed conversion ratios Aquaculture 107:313–332. were being monitored. We anticipate that release procedures will involve a fish pumping device like Gold, J. R., L. B. Stewart, and R. Ward. 2003. Population those widely used in the aquaculture industry for structure of the spotted seatrout (Cynoscion nebulosus) stocking and harvesting netpens. along the Texas gulf coast, as revealed by genetic analysis. In S. A. Bortone (ed.), Biology of the spotted seatrout, p. Seatrout are known to vary genetically among 17–29. CRC Press, Boca Raton, Florida. localities (Gold et al. 2003, Wiley and Chapman 2003), thus, the test group of juveniles from Texas Kestemont, P., S. Jourdan, M. Houbart, C. Melard, M. could not have been released in Mississippi. A Paspatis, P. Fontaine, A. Cuvier, M. Kentouri, and E. project to assess genetic differences among fish from Baras. 2003. Size heterogeneity, cannibalism, and different localities in Mississippi is in development to competition in cultured predatory fish larvae: Biotic and provide information critical to the development of a abiotic influences. Aquaculture 227:333–356. sound release strategy. Until that information is available, we restrict ourselves to releasing juveniles Leber, K. M. 1995. Significance of fish size-at-release on only in the area from which the broodstock were enhancement of striped mullet fisheries in Hawaii. J. World Aquacult. Soc. 26:143–153. acquired. Initial issues addressed by the release will include survival after release, percent recovery, and New, M. B. 1997. Aquaculture and the capture fisheries: movement. More complicated issues such as the Balancing the scales. World Aquacult. 28(2):11–30. effect of size-at-release and time of release will be addressed in the future. Ogle, J. T., and J. M. Lotz. 2006. Characterization of an experimental indoor larval production system for red snapper. N. Am. J. Aquacult. 68:86–91.

Pew Oceans Commission. 2003. America’s living oceans: Charting a course for sea change, recommendations for a new ocean policy, May 2003.

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8 Blaylock Svasand, T., K. E. Jorstad, and T. S. Kristiansen. 1990. Tsukamoto, K., H. Kuwada, J. Hirokawa, M. Oya, S. Enhancement studies of coastal cod in western Norway, Sekiya, H. Fujimoto, and K. Imaizumi. 1989. Size- part 1, recruitment of wild and reared cod to a local dependent mortality of red sea bream, Pagrus major, spawning stock. J. Cons. Int. Explor. Mer. 47:5–12. juveniles released with fluorescent otolith-tags in News Bay, Japan. J. Fish Biol. 35(Supplement A):59–69. Taniguchi, A. K. 1981. Survival and growth of larval spotted seatrout (Cynoscion nebulosus) in relation to Tucker, J. W., Jr. 1988. Growth of juvenile spotted temperature, prey abundance, and stocking densities. seatrout on dry feeds. Prog. Fish-Cult. 50:39–41. Rapp. P-V Reun. J. Cons. Int. Explor. Mer. 178:507–508. Wiley, B. A., and R. W. Chapman. 2003. Population Tsukamoto, K. 1993. Marine fisheries enhancement in structure of the spotted seatrout, Cynoscion nebulosus, Japan and the quality of fish for release. Eur. Aquacult. along the Atlantic coast of the United States. In S. A. Soc. Spec. Publ. 19:556. Bortone (ed.), Biology of the spotted seatrout, p. 31–40. CRC Press, Boca Raton, Florida.

Blaylock 9

Disease Surveillance in Wild and Cultured Stocks of White Seabass (Atractoscion nobilis)

Mark S. Okihiro California Department of Fish and Game 4065 Oceanside Blvd., Suite G Oceanside, California 92056 [email protected]

Keywords: white seabass, Atractoscion nobilis, fish, infectious, disease, survey, aquaculture

Abstract Success of the Ocean Resources Enhancement and Hatchery Program (OREHP) in California has been due, in part, to a comprehensive disease surveillance program geared toward rapid detection of a wide range of infectious and parasitic diseases afflicting both wild and cultured stocks of white seabass (Atractoscion nobilis). Isolation and identification of white seabass (WSB) pathogens is accomplished via a number of proven diagnostic methodologies including necropsy, cytology, histology, electron microscopy, microbiology, serology, and polymerase chain reaction assays. For WSB, major pathogens of concern are: viral nervous necrosis virus (VNNV), Piscirickettsia salmonis, and an as yet uncharacterized herpes virus. Use of an effective disease surveillance program has helped ensure that hatchery epizootics are detected in a timely fashion, and that treatment is appropriate and efficacious. Since OREHP is a stock enhancement program, there is an additional goal of minimizing disease transfer from cultured to wild fish. Disease outbreaks are inevitable with intensive culture; the key is determining which pathogens pose acceptable risks to wild stocks. The most dangerous pathogens are lethal and highly contagious; the worst are novel pathogens to which wild WSB have no immunity. Avoiding introduction of novel pathogens requires determining which diseases occur naturally. A broad sampling of wild populations can determine normal pathogen load and exposure level. The most efficient method of wild fish evaluation is to determine serum antibody levels to specific pathogens using enzyme-linked immunosorbent assays (ELISAs). ELISAs have allowed OREHP to make informed decisions regarding enhancement efforts in California. ELISA results have demonstrated that exposure to VNNV is widespread among wild WSB. Those data give OREHP the option of releasing VNNV-exposed, but healthy, WSB without fear of unleashing a new plague into native stocks. In contrast, exposure to P. salmonis has not been conclusively demonstrated among wild fish, and that novel disease requires a strict discover-and-euthanize approach when found in cultured fish.

Introduction White seabass (Atractoscion nobilis) belong to In response to the collapse of wild stocks, the the family Scianidae and are one of the largest Ocean Resources Enhancement and Hatchery croaker species found in the Pacific Ocean. The Program (OREHP) was formed in 1983. OREHP is a primary host range is from Point Conception, in cooperative effort between the California Department Central California, south through Baja, Mexico. of Fish and Game (CADFG), Hubbs-SeaWorld Historically, the fish has been found as far north as Research Institute (HSWRI), several California Alaska (Eschmeyer and Herald 1983). White seabass public utility companies, and a number of volunteer (WSB) have a maximum size of approximately 40 kg organizations. The primary goal of OREHP is to and have an estimated lifespan of more than 30 years. help restore marine fish populations in California WSB are a popular commercial and recreational fish waters. OREHP works actively to culture several species, with peak catches of 50,000 to 65,000 fish marine finfish species, but the major focus of per year from sport boats (commercial passenger remediation efforts is on WSB; WSB are currently fishing vessels) alone in the late 1940s. Beginning in the only cultured marine fish being released into the early 1950s, catches from both recreational and California waters. As part of the OREHP program, a commercial fishermen started to decline dramatically. dedicated marine fish hatchery was constructed in The fishery was essentially nonexistent by the late 1995. The Leon Raymond Hubbard Jr. Marine Fish 1970s. Hatchery (also known as Carlsbad Hatchery), located

Okihiro 11 in Carlsbad, California, has produced more than The third arm of OREHP’s health assessment 100,000 juvenile WSB for release into the Pacific in and disease prevention program is an ongoing effort each of the past five years. The record year was 2004 to survey wild stocks of marine fish species—with when the hatchery released more than 270,000 fish, special emphasis on those species targeted for either directly from the hatchery, or from one of 14 mitigation or aquaculture. The goal of this disease growout/netpen facilities located throughout assessment survey is to determine which pathogens Southern California. and diseases are naturally occurring among wild The release number for 2005 was expected to be marine fish stocks. This baseline information is only half that of 2004. The primary reason for the critical if we are to avoid releasing lethal pathogens reduced number is that 2005 was one of the worst into potentially naïve populations of wild marine fish. years for diseases since the hatchery was constructed. Flexibacter maritimus and disseminated Vibrio Finding a Competent Fish Pathologist epizootics occurred at the hatchery and four netpen One of the most important steps in any sites. Piscirickettsiosis, which is a lethal rickettsial aquaculture venture or finfish enhancement program disease caused by viral nervous necrosis virus is locating a qualified and experienced fish (VNNV) (Piscirickettsia salmonis), was confirmed at pathologist. Fish pathologists can have either a one netpen site and was investigated at another. Gill veterinary or fisheries background, but should be flukes (Anchoromicrocotyle guaymensis) plagued qualified in the diverse array of subjects required for four netpen sites. As of November 2005, there was a disease prevention, detection, control, and severe herpes virus outbreak at the Carlsbad Hatchery management. Some of the required fields of involving more than 50,000 WSB. There is no single expertise (or at least familiarity) include: general reason for the increased number and severity of biology, marine biology, fish diseases, general disease outbreaks that year, but the 2004–2005 winter pathology, fish pathology, cytology, hematology, was one of the wettest on record for California, and serology, histology, histopathology, general anatomy, water quality early in 2005 was marginal at best with fish anatomy, and molecular biology. Some wide fluctuations in temperature, salinity, and background in toxicology, microbiology, fish particulate loads. Although no specific testing was physiology, surgery, and genetics is also helpful. done, contaminant loads (i.e., xenobiotics and While finding a fish pathologist with a bacteria) from rainwater runoff were assumed to be comprehensive and diverse educational background high throughout Southern California. is important, equally important is finding one with Disease outbreaks are an unfortunate, and extensive practical experience with a variety of fish sometime inevitable, consequence of almost all high- diseases (infectious and noninfectious) in a variety of density marine aquaculture and finfish enhancement fish species. Ideally, experience should be with large programs. To maximize mitigation efforts, while at groups of fish (i.e., herd health) and with managing the same time minimizing disease impacts to both problems associated with outbreaks of highly cultured and wild fish, OREHP uses a three pronged contagious diseases among high-density populations. approach: 1) a comprehensive biosecurity program, The ideal fish pathologist would be one who is 2) health inspections of cultured fish, and 3) wild fish already familiar with the particular fish species in disease surveillance. Biosecurity protocols are use. However, because many remediation efforts geared toward disease prevention and limiting the involve marine fish species being cultured for the spread and impact of diseases when they occur. first time, this is usually not possible. The next best Biosecurity is a key component of any aquaculture approach is to hire a person with sufficient flexibility operation, but is not discussed here. that he or she can adapt his or her training and Health inspections are the second component of experience to the species at hand and to the unique OREHP’s overall disease prevention progam; all disease problems associated with that species. cultured fish are thoroughly evaluated prior to Eventually, given sufficient time and examination of release. At a minimum, cultured WSB are inspected a sufficient number (i.e., hundreds to thousands) of twice. The initial health check is done at the hatchery diseased fish, almost any qualified fish disease prior to transport to net pen facilities; the second specialist can become experienced with a given fish check is done at the net pens just prior to fish being species. released into the Pacific. Additional health Once the appropriate fish disease expert is assessments are done as needed if fish are behaving located and gains experience with the particular fish abnormally or if there is an increase in daily species being cultured, the next major hurdle is to mortality. 12 Okihiro ensure that this disease knowledge base (often growout facilities have logbooks, which can be painstakingly acquired over a period of years) is not accessed to determine if pen maintenance was lost when the fish pathologist leaves, retires, or gets consistent and if there were problems with either eaten by a shark while diving. Knowledge transfer to mechanical systems or volunteers. the next pathologist can be simplified by 1) having a Water quality is the final item to check. Major comprehensive and detailed file of pathology reports, water quality parameters that can affect fish health 2) having detailed written protocols for most (if not and influence disease outbreaks are: 1) temperature, all) of the tasks involved with disease diagnostics, 2) dissolved oxygen, 3) xenobiotic contaminant prevention, and control, and 3) having an extensive spills, and 4) red tide events (dinoflagellate plankton photographic record for all of the major diseases blooms). Marked temperature fluctuations (those encountered in that particular species. Photos should >3°C) that occur over a short period of time (hours to include gross photos of necropsy findings; wet mount days) often precede outbreaks of infectious disease. cytology photos, light micrographs of histologic lesions, as well as transmission electron micrographs Clinical assessment. Knowledge of normal behavior of ultrastructural lesions and pathogens. A for the fish species, age group, and enclosure is comprehensive and well-organized photographic important if deviations from the norm are to be record of diseases, for a given fish species, will go a detected. With WSB, it is important to know how the long way in bridging the knowledge gap for the next different age groups (larvae, juvenile, subadult, and fish pathologist. adult) act when they are in round versus rectangular tanks, concrete raceways, fiberglass raceways, or Diagnostic Approach netpens. Do fish school? At what age do they The diagnostic approach used by OREHP school? In what types of enclosures do they school? includes five major components: 1) history and Are fish normally up near the surface, in the middle background information, 2) clinical assessment of of the water column, or near the bottom? Answers to live fish, 3) necropsy and gross examination, 4) all of these questions are important to define what is sample selection, storage and transport, and 5) normal behavior for a given species, age group, and sample analyses using a variety of diagnostic assays. tank setup. This knowledge can only be acquired over time, so it is always important to take the time to History and background information. Obtaining a observe living fish in both sickness and health. Some thorough history is the first step in obtaining a general clinical signs to look for in unhealthy fish reliable diagnosis. Background information should are: 1) weight loss (head out of proportion to the include specifics on the species, age, size, and body), 2) anorexia (sick fish rarely feed), 3) lethargy number of fish involved. If the fish are at a growout (sick fish swim slowly) and absent or reduced escape facility, it is important to determine from what part of reflex, 4) weakness (sick fish are pushed towards end the hatchery the fish originated and how they were screens or pulled towards drains), and 5) abnormal transported. The number of transport trips that were pigmentation (sick fish are usually darker than needed, as well as any problems encountered during normal). transport, are also important pieces of information. In addition to these nonspecific signs of disease, Daily mortality (both raw numbers and percentage of there are other more specific clinical indicators of ill the population) and mortality patterns over the health. With WSB, even heavily parasitized fish preceding two weeks are important indicators of rarely flash (i.e., attempt to scratch themselves on health, as are feeding rates and patterns. Some fixed objects). Still, when flashing is observed, it is OREHP pen operators feed by hand, which gives wise to check for external parasites. Terminal them a good opportunity to observe their fish, assess spinning or spiraling in juvenile WSB is usually feeding rates, and evaluate overall condition and associated with herpes virus gastroenteritis. A health. Information with respect to tank or pen variety of cutaneous and ocular lesions can also be maintenance is also helpful. Breakdown of observed among a group of fish and will help indicate mechanical equipment can obviously impact water which fish should be selected for necropsy and which quality and fish health. Consistent vacuuming of diseases are most likely to be encountered. For tank bottoms and cleaning of end screens are the example, with WSB, mottled skin pigmentation is primary maintenance items to look for. Inconsistent often indicative of Ichthyobodo infestation, while care—because of personnel shortages or sickness— focal white areas of depigmentation are characteristic should also be looked for and noted. The majority of of P. salmonis. There is never 100% uniformity with respect to the clinical presentation, but with careful Okihiro 13 observation, patterns will usually emerge and will For the gross internal exam, the fish is placed in help indicate from what the majority of fish are right lateral recumbency (i.e., right side down, head suffering. to the left). The entire left abdominal body wall is removed with blunt/blunt tipped scissors to avoid Necropsy. A thorough necropsy is the most puncturing the swim bladder and to avoid lacerating important step in determining why fish are dying. A gonad or liver. Once the abdomen is open, the gonad good necropsy, performed by an experienced is examined to determine sex. Almost all WSB >12 pathologist, will often quickly reveal what disease is cm TL can be sexed grossly. After sex is determined, involved and what the specific etiologic agent is. A the heart, left liver lobe, pyloric cecae, stomach, and well-executed necropsy is also the cheapest, most swim bladder are examined. The left liver lobe and cost efficient means of making the right diagnosis. stomach are then reflected ventrally to expose the Of course, necropsy results are only as good as spleen, intestinal tract, right liver lobe, gall bladder, the fish that are examined, so proper selection of the and endocrine pancreas. The last step is to pull the appropriate fish is critical. Live moribund fish are swim bladder away from the spine and to examine always preferable to dead fish, fresh dead is preferred the head, trunk, and tail kidney. to a fish that has been dead for several hours, and a For the majority of cultured WSB, the brain and dead fish several hours old is preferable to one that otoliths are not examined during routine health has been frozen. A good pathologist will always checks. Brains are dissected and sampled only when select his or her own fish to examine because for the hatchery fish exhibit neurologic signs, have suffered uninitiated, all sick fish generally look alike. A good cranial trauma, or when viral nervous necrosis pathologist will select fish for necropsy based on (VNN) is suspected. For wild WSB, otoliths are specific clinical findings after observing fish in situ. routinely sampled for age assessment, and brains are If obvious moribund fish are not available, then frequently removed for VNN evaluation. The fish should be caught from areas where sick fish dissection is made by using a hacksaw with a metal congregate. In concrete raceways, this means toward cutting blade to make a frontal section above the eyes the back, near endscreens. With fiberglass raceways, to access the brain dorsally. If the initial cut is not this means checking the corners, and with circular deep enough, the cranial vault can be further opened tanks with center standpipes and drains, it means with a scalpel or razor blade. The brain is removed checking the middle of the tank. The number of fish by using fine scissors to sever the olfactory lobes, to necropsy is variable, but the OREHP standard is to cranial nerves, and then the spinal cord. Otoliths are sample six fish per group. The number of fish located in the semicircular canals located below the sampled may be as few as four, if the cause of brainstem. disease is obvious, or as many as 400, if there is need for a large number of different diagnostic assays. Sample Collection, Storage, Transport, and Moribund WSB are euthanized with MS222 Analyses (tricaine methane sulfonate). Total length (TL) and OREHP employs a diverse array of diagnostic standard length (SL) are taken in centimeters; methodologies including cytology, histology, determining wet body weight (in grams) is optional. electron microscopy (EM), microbiology, polymerase If a blood sample is required, it is taken prior to or chain reaction (PCR) assays, and enzyme-linked immediately following euthanasia. The gross immunosorbent assays (ELISAs). Proper sample external exam is the next step and involves careful collection and handling, prior to analyses, ensures assessment of the skin, fins, gills, pseudobranch, oral that the maximum amount of information will be cavity, tongue, and eyes. The skin and fins are obtained. checked for irregular pigmentation, erosion, and ulcers. The gills and pseudobranch are assessed for Cytology. Wet mount cytology is used for the color, filament damage, and parasites. The oral diagnosis of many common bacterial and parasitic cavity and tongue are checked for parasites, lesions, infections in WSB. Cytological samples include gill and deformities. Deformities of the maxilla and and skin scrapings and occasional squash mandible are relatively common among some groups preparations used to assess nodular masses. Gill and of WSB. The eyes are evaluated primarily for skin scrapings are collected using a dull scalpel to evidence of emphysema associated with exposure to minimize cutting into deeper tissues; the goal is to gas supersaturation. Following the gross external scrape parasites and bacteria off target surfaces. For exam, scrapings are taken from the gills and skin for gills, samples of filament tissue are collected from cytological assessment of parasite and bacterial loads. 14 Okihiro the first or second gill arch. With skin samples, the Histology is also a faster and cheaper diagnostic base of the left pectoral fin is initially scraped, alternative for some viral infections (e.g., VNN) and followed by a pass along the skin below the dorsal rickettsial pathogens (e.g., P. salmonis). fin. Small (1–2 mm3) samples of skin and gill are For histology to be effective, a key factor is to transferred onto glass slides and spread into a thin obtain samples as quickly as possible after euthanasia layer by applying a glass coverslip. Coverslipped or natural death. Autolysis, from bacterial or host samples are examined immediately via dark field enzymes, and bile, will rapidly degrade organs and light microscopy for parasites and bacteria. tissues, making interpretation difficult or impossible. The usefulness of the exam is dependent on how For WSB, 10% formalin is routinely used as a long the fish has been dead, when the sample was fixative. Samples are handled as carefully as possible taken, and the thickness (between the slide and to minimize crushing artifacts and to preserve normal coverslip) of the sample. Ideally, samples are taken architecture. Samples from larger parenchymal as soon as possible after the fish has died and organs are cut with Teflon-coated razor blades to a examined as soon as possible after the slide has been thickness of 1 cm (or thinner) to ensure rapid and prepared. If a fish has been dead for too long, and thorough fixation. A 10:1 ratio of formalin to tissue the fish remains in water, the offending parasites may is used. Larval fish <1.5 cm TL are fixed whole. For leave the body. If a fish has been dead for too long, larval and small juvenile fish >1.5 cm TL, the and the fish remains out of water, the offending abdominal cavity is opened prior to fixing the entire parasites may be obscured by excess skin or gill fish. For large juvenile and subadult WSB, the gall mucus, or may have died and be difficult to locate. bladder is carefully removed and either discarded or Squash preparations are typically used to fixed in a separate container. examine focal nodular lesions in parenchymal organs Formalin-fixed tissues are trimmed with a razor such as liver or kidney. Squash preps are also used to blade, placed in 4.0 cm by 2.8 cm plastic cassettes, determine etiologies of ocular infections. Squash and submitted to a commercial laboratory (Central preps are made by excising a small (1 mm3) piece of Histology Facility in Sacramento, California) for the mass, with a scalpel and crushing it between a routine paraffin processing. Tissue sponges are used glass slide and coverslip. For dry lesions, a small with smaller larvae to prevent fish from slipping drop of clean seawater can be included prior to through slots in cassettes and being lost during adding the coverslip. processing. Embedded tissues are sectioned at 5–7 Both squash preps and skin scrapings are µm; sections are mounted on glass slides and stained examined via light microscopy using a standard with hematoxylin and eosin (HE). All HE slides are binocular microscope (Nikon Labophot) and dark evaluated in house by the OREHP fish pathologist. field/phase contrast condenser. For WSB, the dark Slides are assessed to determine which organs are field condenser is almost exclusively used for wet affected, number and types of lesions, lesion severity, mount preparations. The best wet mount and presence of microbial pathogens. If etiologic preparations are thin (<30 µm between the slide and agents are not readily apparent, special stains may be coverslip). Thin preparations allow clear ordered and HE slides may be sent out for review by visualization of bacteria and smaller protozoa. other fish pathologists. Thicker preparations (e.g., those with large scales or particulates) are suitable for large parasites (e.g., Electron microscopy. EM is used to confirm the flukes), but make identifying bacteria difficult or identity of known WSB viral and rickettsial impossible. The majority of marine finfish pathogens pathogens, and to help identify and classify new are motile, so examining slides quickly will allow microbial pathogens. For transmission electron location and identification of parasites by focusing microscopy (TEM), samples are taken from freshly the search on movement. euthanized fish. TEM samples are trimmed to 1 mm3 cubes with Teflon-coated razor blades, and Histology. Histology is employed when necropsy immersion fixed in chilled Karnovsky’s solution. and or cytology fails to provide a diagnosis, or when Fixed samples are stored at 4°C prior to processing. confirmation of a gross diagnosis is required or In cases where samples were not fixed in desired. Histology allows the extent of tissue damage Karnovsky’s, TEM samples can be obtained from to be determined and results in a permanent record either formalin-fixed material or from paraffin for a disease or pathogen. Histology has good histology blocks. Formalin-fixed material is application for larval fish as almost all major organs, transferred to Karnovsky’s, while paraffin-processed for multiple fish, can be examined on a single slide. material is excised from blocks using scalpel blades, Okihiro 15 and deparaffinized prior to processing. Karnovsky’s- Samples are ideally taken from freshly fixed tissues are processed and embedded in epoxy euthanized fish, but can be taken from euthanized or resin, sectioned at 1 µm, and stained with toluidine dead fish that have been stored at 4°C (or on ice) for blue. Following initial screening of toluidine blue up to 12 hours. Sterile technique, employing flame stained sections, ultrathin (800–900Å) sections are sterilized metal loops or prepackaged sterile plastic cut with a diamond knife, mounted on copper grids, loops, is used to take initial samples. Flexibacter is and stained with 2% uranyl acetate and Reynolds’ cultured on specialized cytophaga agar, while Vibrio lead citrate. species are cultured on blood agar. Colonies take 1– Some TEM assessment is done by the OREHP 3 days to appear. The hatchery manager, Paul Curtis, pathologist, but the majority of scope work, and all of routinely processes fungal and bacterial samples at the processing and sectioning, is done by the the Carlsbad Hatchery. Some Vibrio samples have California Animal Health and Food Safety (CAHFS) been sent to the University of California at Davis laboratory in Davis, California. Mounted ultrathin (UC Davis) for speciation. sections are viewed with either a Zeiss or Phillips The major rickettsial pathogen of concern for transmission electron microscope. Cells from target WSB is P. salmonis. The primary target organ for P. organs are examined for subcellular lesions and the salmonis is the liver and lesions appear as multifocal, presence of viral particles in the nucleus or pale tan to yellow granulomas. P. salmonis requires cytoplasm. For nonviral pathogens, lesions are culture on a chinook salmon (Oncorhynchus scanned to locate, identify, and characterize tshawytscha) embryo cell line (CHSE-214) and all rickettsial or protozoan parasites. liver samples from suspect fish are shipped on ice Samples for direct EM examination are used to overnight to UC Davis. identify gastrointestinal (GI) parasites that are shed There are three viral pathogens of concern for into the lumen of the GI tract. Direct EM is cultured WSB: viral nervous necrosis virus (VNNV), specifically used by OREHP to try and identify a viral hemorrhagic septicemia virus (VHSV) and a suspect herpes viral pathogen. Direct EM samples suspect herpes virus that has only been identified are taken from pyloric cecae and intestine; the using TEM. Samples for virus isolation (VI) assays stomach is avoided to prevent sample degradation include: eye and brain (for VNNV), spleen and from gastric acids. The gall bladder (closely kidney (for VHSV) and pyloric cecae and intestine associated with the intestine in WSB) is similarly (for herpes virus). The liver has also occasionally avoided to prevent damage from bile salts. Contents been sampled for VI. With liver, the left lobe is of pyloric cecae or intestine are emptied into plastic collected (the right lobe and associated gall bladder cryovials using forceps, and are frozen and stored at are not sampled). Splenic samples are taken by –70º to –80ºC. Direct EM samples are shipped on freeing the organ from the gastrosplenic ligament dry ice to the CAHFS laboratory. Samples are rinsed (mesentery), being careful not to incise the gall and filtered prior to EM examination for viral bladder. Trunk and tail kidney are sampled by sharp particles. dissection, using either a scalpel or razor blade to free the organ from the attached spine. Eyes are dissected Microbiology. Microbiology includes the fields of by grasping conjuctival tissues with forceps, applying mycology, bacteriology, and virology. All are geared traction and using scissors to cut periocular towards isolation and growth of microorganisms on connective tissue, extraocular muscles, and the optic artificial media or in cell culture. Fungal infections nerve. The dissection and removal of the brain, in cultured WSB are rare. Diagnosis is usually made pyloric cecae, and intestine are covered above. via cytology, but occasionally, fungal colonies will VI samples are stored in chilled (4°C) minimal be isolated on Sabouraud-dextrose agar. Major essential media and shipped overnight to UC Davis bacterial pathogens of WSB are Vibrio spp. and on ice. VNNV is cultured on a snakehead fish cell Flexibacter maritimus. Although infections are also line (SNN-1), while VHSV is cultured on a carp cell routinely diagnosed using wet mount cytology, line (EPC). Both VNNV and VHSV have culture on artificial media is necessary for bacterial characteristic cytopathic effect when grown on the identification and to determine antibiotic sensitivity. appropriate cells lines. Positive cultures are Skin is the primary target organ for both Vibrio confirmed with either PCR or TEM. Attempts have (usually as a secondary invader) and Flexibacter been made to culture the herpes virus using several (usually as a primary pathogen). Vibrio also occurs fish cell lines, but have to date been unsuccessful. as a disseminated infection, attacking kidney and brain. 16 Okihiro Enzyme-linked immunosorbent assay. ELISAs controlled laboratory exposures or from confirmed have been developed by Dr. Ronald Hedrick’s disease outbreaks among hatchery fish. laboratory at UC Davis to assess pathogen exposure in WSB to three major pathogens: VNNV, VHSV, Polymerase chain reaction. PCR assays have been and P. salmonis. ELISAs work by detecting developed by Dr. Ronald Hedrick’s laboratory at UC antibodies specific for the three pathogens in Davis to detect VNNV, VHSV, and P. salmonis circulating blood. Although ELISAs detect exposure infection in WSB. PCR is a sensitive, highly and not infection, the main advantage is that a fish accurate molecular assay designed to detect pathogen could have been exposed months or possibly years genetic material (RNA or DNA) via use of specific prior to sampling and still have detectable antibody primers followed by exponential gene amplification. levels in peripheral blood. The primary drawbacks are: 1) the assays cannot For WSB <15 cm TL, a 1 ml syringe with a 2.5 determine level of infection, 2) the assays cannot cm, 26 gauge needle is used to draw blood. The determine degree of host damage, and 3) a relatively needle is inserted on the ventral midline, adjacent to slow turnaround time. the anal fin, to access the large vertebral vein located ventral to the spinal column. Approximately 0.3–1.0 Diseases and Pathogens of Cultured White ml of blood can be obtained from a single fish. Seabass Blood from 2–6 small fish is often pooled for a single Diseases of cultured WSB can be divided into serum sample. For larger juvenile WSB, a 2.5 or noninfectious, infectious, and parasitic. 3.75 cm 18 gauge Becton Dickinson Vacutainer Noninfectious diseases are not covered here. needle (and associated Vacutainer sleeve) is used to Infectious diseases include viral, bacterial, rickettsial, draw blood. One to 10 ml of blood can be collected and fungal diseases. There are three major viral from a larger juvenile or subadult fish. Blood diseases of concern for cultured WSB: VNN, samples are collected in Vacutainer SST serum VHSV, and herpes virus gastroenteritis (HVGE). separation tubes. Blood is allowed to clot at room VNN is a lethal, highly contagious disease of larval temperature for 20–30 minutes and then stored at 4°C WSB; HVGE is a lethal, highly contagious disease of for up to 48 hours. Clotted blood is centrifuged for juvenile WSB. VHSV has not been confirmed in 6–10 minutes at 1,000–1,300 RCF (g) in a swing WSB, but is of concern because it has been isolated bucket centrifuge. Serum is pipetted off and stored from several baitfish species caught in Southern as 0.5–1.0 ml aliquots in 2 ml plastic cryovials. California waters (Hedrick et al. 2003). Bacterial Serum is frozen and stored at –70º to –80°C. Frozen diseases include cutaneous infections by F. maritimus serum samples are shipped on dry ice to UC Davis and various Vibrio species. In addition, at least one for final ELISA assessment. Vibrio species (tentatively identified as V. ELISAs are layered assays conducted in alginolyticus) is the causative agent of a lethal multiwell plastic plates. The initial antigen layer is disseminated infection involving kidney and brain. derived from viral or rickettsial pathogens grown in Two rickettsial diseases afflict WSB: one is a cell culture. The second layer is serum from wild or transient, benign infection of the gills by an cultured WSB that has varying concentrations of Epitheliocystis sp.; the other, piscirickettsiosis, is a antibodies specific to the pathogen antigens. The lethal systemic infection caused by P. salmonis. third layer is a biotinylated-rabbit anti-WSB Fungal infections, as stated above, are rare. polyclonal antibody. The rabbit antibodies bind Parasitic diseases of cultured WSB include specifically to the WSB immunoglobulin. The fourth metazoan, protozoan, and crustacean parasites. No layer is a horseradish peroxidase (HRP) streptavidin, cestodes have been recovered from cultured WSB, which binds selectively to biotin on the rabbit but three trematode species have been found in gills antibodies. The final layer is tetramethylbenzidine, a of netpen fish: Anchoromicrocotyle guaymensis substrate specific for the HRP enzyme. Positive (previously identified as Cynoscionicola samples change color from yellow to blue. Degree of psuedoheterocantha), Gyrodactylus sp., and an optical density (OD) is determined by an automated unidentified fluke species. Protozoan parasites plate reader. Degree of OD is dependent on the include: three ciliates (Uronema marinum, amount of pathogen-specific WSB antibody present Trichodina sp., and at least one unidentified ciliate), in the serum sample; the greater the OD, the higher four flagellates (Icthyobodo sp., Cryptobia sp., the antibody titer. Positive samples are those with Hexamita sp., and one unidentified flagellated OD higher than that of positive-control samples. protozoan), and one sporozoan (a probable Positive-control serum is obtained from either Okihiro 17 myxosporidian parasite commonly found in and an absence of nucleoli. The diagnosis is mesonephric ducts of WSB in growout pens). confirmed with TEM. There is no PCR assay Parasitic isopods have occasionally been observed available and the virus has not been successfully attached to fins of WSB at one netpen site in grown in cell culture. There is no treatment. Huntington Beach. The three most important diseases of cultured Piscirickettsiosis. Piscirickettsiosis is the third WSB are VNN, HVGE, and piscirickettsiosis. All major disease of concern for OREHP (Arkush et al. three are lethal, highly contagious, and, for the most 2005, Chen et al. 2000). The causative agent is a part, untreatable. As such, these three diseases pose Piscirickettsia salmonis. P. salmonis epizootics have the greatest challenge to OREHP, both from a occurred twice since the hatchery opened. The first hatchery production standpoint and from a disease outbreak occurred in 1998 and involved multiple risk standpoint to wild fish stocks. hatchery systems as well as several netpen sites. The second outbreak occurred in April 2005 and involved Viral nervous necrosis. VNN is a lethal nodaviral a single netpen site at King Harbor in Redondo disease of larval WSB (Curtis et al. 2001). Prior to Beach. Clinically, P. salmonis infected WSB are the spring of 2003, VNN epizootics were a regular slow moving, moribund fish with scattered, small, occurrence at the Carlsbad Hatchery. Since white foci of depigmentation over the flanks. installation of an ozone water treatment system at the Characteristic gross lesions include: linear streaking hatchery in 2003, there have been no outbreaks. The in gill filaments, a fibrinous necrotic exudate over the etiologic agent is a single-stranded RNA virus. VNV heart, and multifocal, pale tan to yellow, nodular primarily infects larval WSB 20–40 days posthatch; masses in the liver. The diagnosis can be confirmed target organs are retina of the eye, brain, and spinal with histology, culture on CHSE-214 cells, PCR, or cord. Clinically, affected larval fish present as TEM. Although treatment with tetracycline paralyzed fish, floating on their sides at the surface of antibiotics is theoretically possible, treatment has the water. Histologically, target organs have thus far not been attempted. characteristic necrotizing and cystic lesions. Presumptive diagnosis is made via observation of Management of Virulent Disease larval fish with classic clinical signs. Confirmation The mortality rate among even the most virulent of VNN is made with histology, TEM, culture on infectious disease in cultured WSB is never 100%, so SNN-1 cells, and PCR. There is no treatment. the question becomes: what should OREHP do with the thousands of fish that have survived exposure to Herpes virus gastroenteritis. HVGE is a lethal, VNNV, HVGE, and P. salmonis? Disease epizootics highly contagious viral disease of juvenile WSB. It that occur at netpen sites can involve up to 10,000 primarily affects smaller (<15 cm TL), younger (<4 cultured WSB; outbreaks in the hatchery’s raceway months) hatchery seabass exposed to ambient lagoon system can impact up to 60,000 older (3–6 month) waters during fall and winter months. There have juvenile fish. Survivors are often fish into which been two confirmed outbreaks and both occurred at OREHP has invested a significant amount of time, the Carlsbad Hatchery: one in the fall of 2002, the effort, and money, and which represent a high second in the fall of 2005. Mortality can exceed percentage of total release numbers. If this were an 3,000/day if HVGE occurs simultaneous with aquaculture operation, the answer would be simple: Flexibacter. Typical clinical signs (when as long as there was no danger of pathogen transfer to uncomplicated with Flexibacter) are: 1) high humans, surviving stocks would either be sold or mortality among fish with no cutaneous lesions, 2) eaten. But since this is an enhancement program, yellow-white stringy fecal casts in the water, and 3) OREHP has to consider the relative risk of disease terminal spiraling prior to death. Target organs are transfer to the wild fish stocks they are seeking to stomach, pyloric cecae, and intestine. Gross lesions help. include moderate to massive dilation of the entire GI tract, excessive amounts of clear fluid in the GI tract, Disease Surveillance of Wild WSB occasional gastric intussusception, variable ascites, To assess the risk to wild seabass from lethal, and dilated urinary bladders. Histologically, there is highly contagious diseases occurring in cultured multifocal necrosis of mucosal epithelium of pyloric WSB, OREHP initiated a comprehensive disease cecae and intestine, with minimal inflammation. surveillance program in 2002. The goal is to survey Some epithelial cells may have marginated chromatin wild marine fish populations in order to determine

18 Okihiro which lethal infectious diseases are naturally profiles, but did not contain nucleocapsid material occurring or endemic to wild stocks. The rationale is and was classified as negative for herpes virus. that if a disease is endemic among wild fish, then the relative risk from exposed or infected hatchery fish is Wild Fish Disease Survey Conclusions relatively small. Preliminary results from OREHP’s wild fish Although WSB were the primary focus of wild survey lead to three conclusions. First, VNNV is fish surveys, other native California marine fish probably a naturally occurring disease among wild species in which OREHP is interested were also WSB. Second, it is likely that P. salmonis is an sampled. Some of these other species include: exotic disease. Third, the disease status of wild WSB California sheephead (Semicossyphus pulcher), for VHSV and herpes virus is still unknown. California halibut (Paralichthys californicus), California yellowtail (Seriola lalandi), lingcod Impact on OREHP Policies and Hatchery (Ophiodon elongatus), vermillion rockfish (Sebastes miniatus), bocaccio rockfish (S. paucispinis), and Operations cowcod rockfish (S. levis). OREHP’s plan of action The impact wild fish disease survey data have on was simple: 1) sample a large number of wild fish, 2) hatchery operations is that OREHP can now make assess pathogen exposure and infection rates, and 3) informed decisions regarding what to do when use the data to make informed decisions regarding virulent disease outbreaks occur. With VNNV release of infected or exposed hatchery fish. epizootics, the hatchery can reasonably decide to Total catch numbers after 3.5 years of sampling salvage survivors and release them at a later date were: 272 wild WSB, 124 bocaccio rockfish, 43 when they are healthy. In contrast, with P. salmonis, vermillion rockfish, 6 cowcod rockfish, 146 the assumption has to be that this disease does not California halibut, 83 sheephead, and 17 assorted occur among wild WSB, and that any cultured other fish species. Sample numbers for just wild seabass infected or exposed to this pathogen should WSB were: 195 ELISA serum samples, 100+ brain be euthanized. With herpes and VHSV, there still is samples for VNNV VI and PCR, 100+ eye samples not enough information to determine whether or not for VNNV VI and PCR, 80+ spleens for VHSV VI these two diseases are endemic to wild fish stocks. and PCR, 80+ kidneys for VHSV VI and PCR, 80+ We believe that herpes virus is probably a naturally liver samples for P. salmonis isolation and PCR and occurring disease among wild WSB, but until there is 100+ intestinal content samples for direct EM definitive proof, OREHP policy will be to operate as assessment of herpes virus. All wild fish samples if it too were an exotic disease. were sent to UC Davis and sample analyses are ongoing. References Preliminary ELISA results indicate that VNNV Arkush, K. D., A. M. McBride, H. L. Mendonca, M. S. exposure is widespread among wild WSB. Among Okihiro, K. B. Andree, S. Marshall, V. Henriquez, and R. P. Hedrick. 2000. Genetic characterization and wild juvenile or subadult WSB (i.e., those <72 cm experimental pathogenesis of Piscirickettsia salmonis TL), 18% (14/78) of serum samples were ELISA- isolated from white seabass Atractoscion nobilis. Dis. positive for VNNV exposure. Among wild adult Aquat. Organ. 63:139–149. WSB, 53% (9/17) of serum samples were VNNV- positive with ELISA. In contrast, none of the 94 Chen, M. F., S. Yun, G. D. Marty, T. S. McDowell, M. L. serum samples analyzed thus far via ELISA have House, J. A. Appersen, T. A. Guenther, K. D. Arkush, and been positive for P. salmonis exposure. ELISA R. P. Hedrick. 2000. Piscirickettsia salmonis-like results for VHSV are pending, and currently there is bacterium associated with mortality of white seabass no ELISA assay specific for the herpes virus that Atractoscion nobilis. Dis. Aquat. Organ. 43:117–126. occurs in WSB. Curtis, P. A., M. Drawbridge, T. Iwamoto, T. Nakai, R. P. All attempts to isolate virus or rickettsia from Hedrick, and A. P. Gendron. 2001. Nodavirus infection of wild WSB have failed. A small number of PCR juvenile white seabass, Atractoscion nobilis,cultured in samples have been assessed and these have also southern California: First record of viral nervous necrosis turned up negative for virulent pathogens known to (VNN) in North America. J. Fish Dis. 24:263–271. occur in cultured WSB. Results from the majority of PCR samples, however, are still pending. Of the Eschmeyer, W. N., and E. S. Herald. 1983. Croakers: more than 100 intestinal content samples taken, 50 Family Sciaenidae. In R. T. Peterson (ed.). A field guide have been analyzed via direct EM for herpes virus. A to Pacific Coast fishes of North America, p. 218–222. single direct EM sample had suspect hexagonal Houghton Mifflin Company, Boston, Massachusetts. Okihiro 19 Hedrick, R. P., W. N. Batts, S. Yun, G. S. Traxler, J. Kaufman, and J. R. Winton. 2003. Host and geographic range extensions of the North American strain of viral hemorrhagic septicemia virus. Dis. Aquat. Organ. 55:221– 220.

20 Okihiro Juvenile Mass Production of the Black Rockfish (Sebastes schlegeli) in FRA Miyako Station: Present Status and Problems

Masahiro Nakagawa Miyako Station National Center for Stock Enhancement Fisheries Research Agency 4-9-1, Sakiyama, Miyako Iwate 027-0097, Japan [email protected]

Junichi Adachi Shibushi Station National Center for Stock Enhancement Fisheries Research Agency

Ken Shimizu Fisheries Stock Enhancement Department Fisheries Research Agency

Satoshi Shiozawa Amami Station National Center for Stock Enhancement Fisheries Research Agency

Keywords: Sebastes schlegeli, black rockfish, mass production, stock enhancement

Abstract We reviewed the results of black rockfish (Sebastes schlegeli) mass production at FRA Miyako Station, Japan, between 1982 and 2004. Miyako Station began testing juvenile production of the species in 1980. Initially, live food was mainly used for the tests, but cultivation and securing that food required a large amount of labor. Two types of food, Artemia nauplii and formulated feed, were used for juvenile production beginning in 1999; thus, a juvenile production system that was free from the difficulties involved with the cultivation of most live foods was established. A juvenile black rockfish with a total length of 30 mm currently costs 8.5 yen. The survival rate of juveniles now averages approximately 50%, but it used to fluctuate widely from 19.6% to 81.4%. Early mortality of juveniles; in particular those up to 10 days after birth, was higher and more variable. Even now, early mortality is an obstacle to juvenile production. To stabilize the technique for juvenile production of this species, it is essential to develop a method to curb early mortality.

Introduction Juveniles of six rockfish species (Sebastes (Fisheries Agency & Fisheries Research Agency schlegeli, S. inermis, S.s pachycephalus, S.s vulpes, S. 2004). The species is also important to mariculture thompsoni and S. oblongus) are currently produced in in Korea, as is the Japanese flounder (Paralichthys Japan. Of those species, the black rockfish, grows olivaceus) (Lim et al. 2004). fastest (Nagasawa 2001) and moves in a limited A study of the production of juvenile black range after being released (Nakagawa et al. 2004). rockfish was undertaken by the fisheries For those reasons, in 2002, 3.5 million juvenile black experimental station of Aomori Prefecture in the rockfish—70% of all rockfish produced that year 1970s. That was followed by production of juveniles were reared for stock enhancement and mariculture of the species in Hokkaido, Akita, Yamagata, Miyagi, Hiroshima, Fukushima, Niigata and Ehime Nakagawa 21 Prefectures (Kusakari 1995). The Japan Sea-Farming Second period (1994–1998). An automatic Association (JASFA, predecessor of the Miyako feeding machine was developed that was capable of Station of the National Center for Stock dispensing a formula feed consisting of minute Enhancement, Fisheries Research Agency) began particles (400 µm), making it possible to employ it testing mass production of juveniles in 1980. The early in life the fish. Rearing during the period began technique for juvenile production is now considered by providing a combination of three types of feed: to be almost established, but the scale of rearing rotifers, Artemia nauplii and formula feed. The tanks varies among research agencies. Development production system, which had been centered on live of the juvenile production technique at Miyako feed, was changed to one based on formula feed, Station in recent years has therefore been aimed at thereby saving labor. reducing the unit cost of juvenile production. On the other hand, the survival rate of juveniles with total Third period (1999–present). It was thought lengths up to 30 mm has now reached approximately possible to start using Artemia nauplii as an early live 50%, but it has varied greatly from 19.6% to 81.4% food because the average total length of newborn (Shiozawa 1985, 1986, 1988; Shimizu 1989, 1990, larvae of the black rockfish is 7 mm. Rotifers and 1991, 1992; Adachi 1993, 1994, 1995, 1996, 1997; Artemia nauplii had been used previously in Nakagawa 1998, 1999a, 2000, 2001, 2002, 2003). combination as live food for juvenile production, but This cannot be described as a stable survival rate. it was suggested that juvenile production was This report reviews results of the examination of possible by feeding Artemia nauplii alone (Nakagawa juvenile production at Miyako Station. 2000). To test the hypothesis black rockfish larvae Subsequently, we describe the tasks that remain to be were fed either a combination of live rotifers and accomplished. Artemia nauplii (Group I) or Artemia nauplii alone (Group II). Larvae were reared for 15 days in 500 L Changes in Feed Composition tanks. The average total length ±SD after 15 days of Table 1 shows changes in the types of feed given rearing was 12.4 mm ±1.1 mm in Group I larvae and to larvae and early juvenile black rockfish. 12.6 mm ±0.7 mm in Group II. No significant difference (p > 0.05) was found between the two Table 1. Changes in feed composition on mass production of groups. The average survival rates were 65.1% in black rockfish, Sebastes schlegeli. Group I and 64.9% in Group II. Therefore, two types of feed, Artemia nauplii and formula feed, were used for mass production of black rockfish beginning in 1999. A juvenile production system that does not require rotifer cultivation was thus established.

Current Rearing Conditions Used for Mass Production For juvenile production, it is most important to ensure that few individual larvae are deformed or swim with marked instability. The larvae were quantified, and healthy larvae were transferred using a 50 mm diameter siphon hose to 50,000 L square First period (1982–1993). Juveniles were concrete tanks (8×4.8×1.6 m) at a density of 10 produced by giving live foods such as rotifers larvae/1 L. For production of juvenile black rockfish, (Brachionus plicatilis), Artemia nauplii, cultured we used a titanium and steel heater that used heat Altemia, eggs of pollock (Theragra chalcogramma) from a boiler, a ventilation device with a blower, an and Japanese flounder, frozen larvae of black automatic bottom vacuum cleaner and an automatic rockfish, frozen water fleas and frozen mysids. feeder for formula feed. Artemia nauplii at 5–300 Dependence on live food was high and a large million per tank were provided in two or three amount of labor was required to cultivate and secure feedings each day. Incubation of Artemia eggs was feed during the period. The spread of disease and controlled at a tank temperature of 22°C and deterioration of rearing water were feared because eggshells and Artemia nauplii were separated 42 h fish eggs were used as food. after hatching. Artemia nauplii were enriched for 24 h at 20°C by addition of nutrients (Plus-Aqualan, 22 Nakagawa BASF Japan Ltd.) at a density of 50 g/1,000 L. One certification method, but no significant differences liter of freshwater chlorella concentrate (Chlorella were found among periods (p > 0.05). Therefore, it sp., Fresh Green 600, Nisshin Science Co., Ltd.) was was concluded that changes in feed composition did added to the rearing water in two or three portions a not cause a decline in survival rate. Labor savings in day for up to 30 days after birth (DAB). Formula juvenile production and improvement in production feed was given from about 10 DAB in amounts of efficiency became possible through a reduction in the 50–5,000 g per tank in 8–16 portions daily. The variety and type of feed required for juvenile water temperature was increased by 1ºC/day from production. 13°C at the beginning of rearing period to the optimum rearing temperature of 18ºC, after which it Table 2. Results of mass production breeding experiments in was maintained at that temperature. The daily water the black rockfish, Sebastes schlegeli. exchange rate was set at 50–100% of the total from 0–10 DAB, 100–300% from 10–20 DAB, 300–800% from 20-30 DAB, and 800–1,200% after 30 DAB. Sand-filtered seawater was used for rearing larvae and early juveniles, and for incubation and nutrient enrichment of Artemia nauplii. The tank floors were cleaned using an automatic bottom vacuum cleaner every day and dead larvae were counted at that time. The total length of the larvae or juveniles was measured every five DAB, and specimens (fixed in 10% formalin) were collected to determine their developmental stages.

Survival Rate and Unit Number of Juveniles Produced Table 2 presents the results of juvenile production examinations that were conducted using

50,000 L tanks. The number of juveniles produced represents the sum of the number of juveniles for each 1,000 L of rearing tank (hereafter called the unit Growth and Development production number) because the number of tanks Figure 1 shows the growth of larvae and juveniles reared in 50,000 L tanks. The equation Y= used varied among years. As indicated in the 0.184x 2 classification above, 1982–1993 was defined as the 5.6609e (r = 0.9967) represents the relationship first period, 1994–1998 constituted the second between the total length and DAB. Variations period, and 1999 and after was the third period. The became greater as rearing progressed. unit production numbers of juveniles, rearing times, Table 3 shows results of observations of the and the total lengths of juveniles at the end of developmental stages of larvae and juveniles reared production covering each of three periods were in 50,000 L tanks. Larval development was compared using Scheffé’s F certification method. classified into seven stages according to the method Results showed no significant differences among the of Nagasawa (2001): A preflexion larva, B flexion periods in the unit production numbers of juveniles larva I, C flexion larva II, D postflexion larva, (p > 0.05), but the total lengths of juveniles at the end E transforming larva, F pelagic juvenile I and G of production and the rearing times differed pelagic juvenile II. significantly between the first and third periods Larvae that reached stage B immediately after (p < 0.05). An increase in mortality rates as a result birth were observed in 2001 and 2003. Tanks in of handling at harvest was observed when the total which the larvae were born had only small capacities length of juveniles averaged 25 mm at the end of (1,000 L). Therefore, the possibility of larvae born production. The significantly prolonged rearing the previous day or earlier remaining in the tank was times were attributable to the increased size of the considered extremely low. Therefore, it was assumed harvest, which was intended to raise the survival rate that the larvae that reached stage B immediately after after harvesting. Survival rates were converted into birth had overgrown in the mother’s body. This variables and compared using Scheffé’s F seems to have resulted from a time gap between the growth of the larvae and their delivery, but its Nakagawa 23 mechanism and causes remain unknown. It took Mortality Patterns about 5 days for larvae to move between stages from The survival rate of black rockfish at Miyako A to D, but 10 days were required from stage D to E Station reached nearly 50%, but it remained unstable, and more than 15 days from stage E to F. The later fluctuating widely from 19.6% to 81.4% (Table 1). the stage, the more days were required to develop to Daily changes in the mortality rate during the rearing the next stage. In all years except 1999, the larvae time (Figure 2) and in the shapes of individuals were reached developmental stage G 40 DAB. observed during juvenile production from 1998–2003 to accurately determine mortality patterns in early juveniles. Although minor differences were visible, depending on the rearing lot, increases in the mortality rate were observed in larvae and juveniles from 0–10 DAB, 18–22 DAB and 30–35 DAB, a pattern that was common to all years. It is noteworthy that a mortality rate amounting to 10% of the total in one day was observed in early juveniles from 3–4 DAB in 2000. Normal larvae at that time had reached developmental stages B–C, and the average total length was 7.7 mm ±0.3 mm. However, dead fish were numerous in developmental stages A– B and their average total length was 6.8 mm ±0.2 mm, or smaller than at birth. Those dead fish were Figure 1. Growth of larvae and early juveniles in black presumed to have died of starvation because they had rockfish, Sebastes schlegeli. Closed circles and vertical lines deformed mouths and vertebrae. Furthermore, every indicate mean total length and one unit of standard deviation fish in that population had an empty stomach. of total length. Between 18 and 22 DAB, both surviving and dead fish reached developmental stage D or E, with no Table 3. Developmental-stage composition (%) in larval and significant difference in average total length. early juvenile Sebastes schlegeli. Numerous food organisms were found in the digestive tracts of fish that died at 18–22 DAB, which differed greatly from the scenario in dead fish found 0–10 DAB. One fish was seen swimming belly-up on the surface of the rearing water the day before the increase in mortality rate. When dissected, that individual was found to have an air bladder that was far more inflated than those of normal fish, implying death from gas-balance disorder. Aerating the seawater immediately before it is poured into the rearing tanks minimizes mortality. Mortalities of larvae from 30 to 35 DAB occurred from cannibalism that was attributed to differences in growth rates.

Unit Cost of Juveniles Based on rearing data for 2003, we calculated the unit cost of juvenile black rockfish reared to a total length of 30 mm. Computed expenses included wages; electricity and heating bills; costs of feed, materials, and consumables; as well as rental costs, but they did not include depreciation costs of the facilities. Unit costs of juveniles were calculated by dividing the costs of the two production processes of broodstock rearing and juvenile production by the number of fish produced. That result was 8.5 yen. Itemization revealed that wages accounted for 39.6%

24 Nakagawa this species mate in November of each year (Mori et al. 2003), but they discharge unfertilized eggs if they do not mate at around the time of spawning. The new technique has also made it possible to use the cannulation method to select broodstock that discharge unfertilized eggs. Consequently, the technique for obtaining larvae intensively is nearly complete. As noted here, however, many individuals among larvae obtained in large quantities are apparently deformed or swim unsteadily, accounting for a major fraction of early mortalities. This phenomenon occurs immediately after spawning. For that reason, it might be attributable to some cause that is inherent in the broodstock. Stabilizing the technique for the Figure 2. Changes in the mortality of black rockfish Sebastes production of juveniles of this species demands schlegeli larvae and early juveniles during 1998–2003. Closed circles indicate mortality. reexamination of the way broodstock are managed. Development of techniques for minimizing early mortality rates is also necessary, as are techniques for of the total, followed by electricity and heating bills identifying abnormal larvae in the embryo stage, at 25.3% and the cost of feed at 15.8%. Those three before delivery. items added up to more than 80% of the total expenses. Unit costs of juveniles by size were References calculated tentatively, including expenses for Adachi, J. 1993. Juvenile production of black rockfish. intermediate rearing as indicated by Nakagawa and Annual report of the Japan Sea-Farming Association in Okouchi (2003a). Results were: 8.5 yen for 30 mm 1991, Tokyo, p.159–162 [in Japanese]. fish, 10.4 yen for 40 mm, 13.5 yen for 60 mm, 15.9 Adachi, J. 1994. Juvenile production of black rockfish. yen for 80 mm, and 18.3 yen for 100 mm. Annual report of the Japan Sea-Farming Association in 1992, Tokyo, p.143–145 [in Japanese]. Future Tasks For efficient juvenile production, it is necessary Adachi, J. 1995. Juvenile production of black rockfish. to develop a technique to secure quality newborn Annual report of the Japan Sea-Farming Association in larvae intensively and in massive quantities. Each 1993, Tokyo, p.153–155 [in Japanese]. day, more than 1 million newborn larvae of multiple- spawning fish such as Japanese flounder are now Adachi, J. 1996. Juvenile production of black rockfish. Annual report of the Japan Sea-Farming Association in obtainable on a stable basis, thanks to advances in the 1994, Tokyo, p.141–143 [in Japanese]. techniques used for broodstock rearing (Thuzaki 1994). However, the black rockfish, which is Adachi, J. 1997. Juvenile production of black rockfish. viviparous, spawns only once a year (Kusakari 1995). Annual report of the Japan Sea-Farming Association in In addition, the larvae produced per broodstock are as 1995, Tokyo, p.165–167 [in Japanese]. few as 200,000 (Nakagawa and Okouchi in press). The fish do not synchronize their spawning in groups Fisheries Agency & Fisheries Research Agency. 2004. (Nakagawa and Hirose 2004). Therefore, it has been Materials on the production and release of fingerlings for difficult for most juvenile production agencies to sea-farming in 2002, Tokyo, p. 431 [in Japanese]. obtain larvae of the species in large quantities at one Kusakari, M. 1995. Studies on the reproductive biology time. Using a cannulation method, Yamada and and artificial juvenile production of kurosoi, Sebastes Kusakari (1991) developed a technique to estimate schlegeli. Bull. Hokkaido Pref. Fish. Exp. Stn. 47:41–124 the birth dates of larvae from examination of [in Japanese with English abstract]. embryonic developmental stage. Using the same technique, Nakagawa and Okouchi (in press) have implemented a method of selecting female broodstock estimated to be near spawning and thereby obtaining larvae intensively. Broodstock of

Nakagawa 25 Lim, S. R., S. M. Choi, X. J. Wang, K. W. Kim, I. S. Shin, Nakagawa, M., H. Okouchi, and J. Adachi. 2004. T. S. Min and S. C. Bai. 2004. Effects of dehulled Stocking effectiveness of black rockfish Sebastes schlegeli soybean meal as a fish meal replacer in diets for fingerling released in Yamada Bay evaluated by a fish market census. and growing Korean rockfish Sebastes schlegeli. In K. M. Leber, S. Kitada, H. L. Blankenship and T. Aquaculture 231:457–468. Svåsand (eds.), Stock enhancement and sea ranching, p. 501–511. Blackwell, Oxford, UK. Mori, H., M. Nakagawa, K. Soyano, and Y. Koya. 2003. Annual reproductive cycle of black rockfish Sebastes Nakagawa, M., and K. Hirose. 2004. Individually specific schlegeli, in captivity. Fish. Sci. 69:908–921. seasonal cycles of embryonic development in cultured broodstock females of the black rockfish, Sebastes Nagasawa, T. 2001. Early life history of rockfishes schlegeli. Aquaculture 233:549–559. (Genus Sebastes) in the Sea of Japan. Bull. Japan Sea Natl. Fish. Res. Inst. 51:1–132 [in Japanese with English Nakagawa, M., and H. Okouchi. In press. Examination of abstract]. embryonic developmental stage by cannulation to synchronize the timing of parturition in black rockfish Nakagawa, M. 1998. Juvenile production of black Sebastes schlegeli broodstock females. Aquacult. Sci. 53 rockfish. Annual report of the Japan Sea-Farming [in Japanese with English abstract]. Association in 1996, Tokyo, p. 160–161 [in Japanese]. Shimizu, K. 1989. Juvenile production of black rockfish. Nakagawa M. 1999a. Juvenile production of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1987, Tokyo, p. 175–176 [in Japanese]. Association in 1997, Tokyo, p. 169–170 [in Japanese]. Shimizu, K. 1990. Juvenile production of black rockfish. Nakagawa, M. 1999b. Bloodstock management of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1988, Tokyo, p. 194–195 [in Japanese]. Association in 1997, Tokyo, p. 34–36 [in Japanese]. Shimizu, K. 1991. Juvenile production of black rockfish. Nakagawa, M. 2000. Juvenile production of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1989, Tokyo, p. 150–151 [in Japanese]. Association in 1998, Tokyo, p. 176–178 [in Japanese]. Shimizu, K. 1992. Juvenile production of black rockfish. Nakagawa, M. 2001. Juvenile production of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1990, Tokyo, p. 181–182 [in Japanese]. Association in 1999, Tokyo, p. 158–159 [in Japanese]. Shiozawa, S. 1985. Juvenile production of black rockfish. Nakagawa, M. 2002. Juvenile production of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1984, Tokyo, p. 157–170 [in Japanese]. Association in 2000, Tokyo, p. 44 [in Japanese]. Shiozawa, S. 1986. Juvenile production of black rockfish. Nakagawa, M. 2003. Juvenile production of black Annual report of the Japan Sea-Farming Association in rockfish. Annual report of the Japan Sea-Farming 1985, Tokyo, p. 178–180 [in Japanese]. Association in 2001, Tokyo, p. 53–54 [in Japanese]. Shiozawa, S. 1988. Juvenile production of black rockfish. Nakagawa, M., and H. Okouchi. 2003a. The effects of Annual report of the Japan Sea-Farming Association in density and feeding frequency on the intermediate culture 1986, Tokyo, p. 191–195 [in Japanese]. of young black rockfish, Sebastes schlegeli. Saibai Giken 30:55–60 [in Japanese with English abstract]. Thuzaki, T. 1994. Juvenile production of Japanese flounder. Annual report of the Japan Sea-Farming Nakagawa, M., and H. Okouchi. 2003b. Abnormal Association in 1996, Tokyo, p. 121–123 [in Japanese]. spawning of female black rockfish, Sebastes schlegeli, in the absence of copulation. Saibai Giken 30:75–77 [in Yamada, J. and M. Kusakari. 1991. Staging and the time Japanese with English abstract]. course of embryonic development in kurosoi, Sebastes schlegeli. Env. Biol. Fish. 30:103–110.

26 Nakagawa Construction of a BAC Library from XY Japanese Flounder Using Frozen Sperm Genomic DNA

Hiroyuki Okamoto and Hiroyuki Nagoya

Eiichi Yamamoto Tottori Prefectural Fisheries Experimental Station Tomari, Tohaku, Tottori 689-0602, Japan

Takashi Sakamoto, Kanako Fuji, and Nobuaki Okamoto Tokyo University of Marine Science and Technology Konan, Minato, Tokyo 108-8477, Japan

Kazuo Araki National Research Institute of Aquaculture Tamaki, Mie 519-0423, Japan

Ichiro Nakayama National Research Institute of Fisheries Science Fukuura, Kanazawa, Yokohama 236-8648, Japan

Keywords: Japanese flounder, Paralichthys olivaceus, bacterial artificial chromosome, cosmid, frozen sperm, heterozygosity

Abstract Progress in genomic breeding for aquaculture requires several molecular genetics tools, which will facilitate analysis of genetic linkage and synteny or to clone genes associated with desirable commercial traits. Such tools include a recombination map, a physical (e.g., chromosome and radiation hybrid) map and genomic (bacterial artificial chromosome [BAC] and cosmid) libraries. We have constructed BAC and cosmid libraries from the frozen sperm of Japanese flounder (Paralichthys olivaceus). The BAC library was generated from an XY male fish, the heterozygosity of which was confirmed by examination of the male:female ratio of its offspring. This BAC library contains DNA sequences from both the X and Y chromosome and will constitute a useful tool for the analysis of Y-specific genes. The sperm had been frozen in liquid nitrogen and stored in a freezer at –80°C for more than one month. This study suggests that frozen fish sperm can be used in the construction of BAC or cosmid libraries even after prolonged storage. We have isolated several clones (e.g., major histocompatibility complex [MHC] class Ia) using the BAC library to analyze the synteny among fish species. Our results further indicate that freezing does not create a bias in the library.

Introduction The Japanese flounder (Paralichthys olivaceus) (390Mb), and one-half of the zebrafish (Danio rerio) constitutes a key species for marine culture in Japan. (1.6Gb) genomes (Uwa and Iwata 1981, Brenner et It has a relatively well understood biology and a al. 1993, Ojima and Yamamoto 1990, Ciudad et al. relatively small genome (approximately 0.71 2002). Therefore, flounder is a good model to pg/haploid [700 Mb]) (Ojima and Yamamoto 1990), understand aquaculture fish and to develop genomic which is shared among 24 chromosomes. This breeding. genome size is estimated as similar to medaka In a project of the Fisheries Research Agency (Oryzias latipes) (850Mb), red sea bream (Pagellus “Genomic Breeding,” we have been producing bogaraveo or Pagrus major) (900Mb), and yellowtail genomic resources for Japanese flounder, including (Seriola quinqueradiata) (800Mb), twice that of fugu bacterial artificial chromosome (BAC) and cosmid (Takifugu spp., among others) libraries. The genomic libraries, having large insert Okamoto 27 DNA, can facilitate rapid construction of a physical 30°C in a block incubator. The tube of frozen sperm map and reduce the cost of structural analysis of large was placed in the block incubator, and 1 ml of chromosomal regions. They can also help us find a preheated Ringers solution was added. The tube was responsible gene for a desirable commercial trait by gently shaken with the sperm pellet. The thawed positional cloning. pellet was poured into the tube of the rest of the The original BAC library of Japanese flounder Ringers solution. The tube was transferred in ice and was constructed using the sperm of an XX gently shaken again to initiate sperm dispersal homozygous cloned fish (Katagiri et al. 2000). without using a pipette. After removing the However, that former BAC library had lost the Y aggregates from the thawed sperm solution, we chromosome, so we proceeded to construct a BAC constructed the agarose plugs with the solution library from an XY heterozygous flounder to get containing 8 × 108 cells/plug. The agarose gel plug complete information from a set of all chromosomes. was constructed as described by Peterson et al. For this purpose we had to keep the sperm frozen for (2000). 7 months until we received the results of a heterozygosity test. In this study, we describe a BAC Construction and size estimation of BAC and library made from a heterozygous flounder using its cosmid library. The BAC library was constructed frozen sperm, and also a cosmid library as a DNA by Geno Tech Co. (Tsukuba, Japan) using the resource from an infectious disease-resistant fish. agarose gel plugs from the frozen sperm of the TY-4 flounder and standard methods. The high molecular Methods and Materials weight DNA in the agarose plug was partially Animals. Japanese flounders for constructing the digested with MboI. After a two-step size selection, BAC libraries were obtained from the Tottori the digested DNA was integrated into the pBAC3.6 Prefectural Fisheries Experimental Station. They vector. were comprised of five pairs of flounder that were The cosmid library was constructed with the mated one-to-one in June 2003, and whose offspring Copy Control Fosmid library kit (Epicentre) from the were reared until January 2004. For constructing the frozen sperm of an anti-Edwardsiella tarda XX cosmid libraries, the flounder was an anti- cloned Japanese flounder established at the Hyogo Edwardsiella XX clone obtained from the Hyogo Prefectural Fisheries Experimental Station in 2002. Prefectural Fisheries Experimental Station. Analyses of insert size of BAC and cosmid clones were performed by pulse field gel Heterozygosity test of male fish. Five male fish electrophoresis (PFGE) using the fragments were designated TY-1 through TY-5 and their generated by NotI digestion containing the insert offspring were subjected to mild estradiol treatment DNAs and BAC or cosmid vector. (10 µg/l) for 2 hours every day from 15 June 2003 to 23 July 2003. The fish were observed from body Results and Discussion lengths of 13–50 mm and from 34 to 72 days after To check the heterozygosity of the male fish, we hatching. A preliminary check of the sex ratio of the used five one-to-one mating pairs in June 2003, and offspring was performed in December 2003 by checked the sex ratio of each offspring 7 months after observing the gonads of the fish. The final check was fertilization. Before checking the sex ratio, the performed in January 2004. offspring were treated with mild estradiol as previously described. The estradiol treatment did not Freezing and defrosting of sperm. Semen from the influence the XY fish to be male and repressed the TY-1 through TY-5 fish was centrifuged in June XX fish from transforming to male because of water 2003 to obtain a sperm pellet. We dispensed 300– temperature. According to the data obtained from 500 µL of semen into a serum tube or a 2 ml checking the sex ratio of the fish two times, the centrifuge tube. The semen was centrifuged for 10 male:female sex ratio of the offspring of the TY-4 minutes at 2,000 g at room temperature (preferably at flounder was 53:47; the numbers of male and female 4°C). The supernatant was carefully removed in ice. offspring were 61 and 54, respectively. The sperm pellet in the tube was immediately frozen We concluded the TY-4 flounder was a XY male in liquid nitrogen. The tube was stored in a deep fish, and proceeded to make a BAC library from the freezer at –80°C until October or December 2003. frozen sperm of TY-4. Because the mating season of To defrost the frozen sperm pellet, we incubated flounder is only during spring, we stored the sperm 5 ml of Egami’s Ringers solution for marine fish at until we could determine the sex ratio of the offspring

28 Okamato several months later. We froze the sperm of the five paternal fish in June 2003. After checking the sex ratio of the offspring, we prepared agarose plugs for constructing a BAC library, and placed an order for a library with Geno Tech Co. using the plugs. We checked that the length of the high molecular weight DNA of the agarose plug was more than 550 kb, which should be further evaluated for constructing the BAC library (Figure 1).

Figure 1. Check length of high-molecular-weight DNA in agarose gel plug.

Figure 2. Analysis of insert size of BAC clones of The BAC library of TY-4 consisted of 110,592 heterozygous Japanese flounder. (A) NotI-digested BAC clones arrayed in 288 microtiter plates with 384 DNAs and vector arm on PFGE. (B) Insert size distribution of the representative BAC clones. wells. Analysis of insert size of the library was performed by PFGE using the fragments of BAC clones, which were produced by NotI digestion. Second, we screened 24 microsatellite markers, The insert size distribution is indicated in Figure which were associated with the 24 linkage groups 2b. The average insert size was calculated to be (Coimbra et al. 2001, 2003). We could isolate at 140.7 kb. From the average insert size of this library least one BAC clone for all 24 linkage markers (data (140.7 kb), the total number of clones (110,592), and not shown). The results showed that freezing the the genomic size of Japanese flounder sperm does not create a chromosome bias. This (approximately 700Mb) (Ojima and Yamamoto study suggests that frozen fish sperm can be used in 1990), we estimated the coverage of our BAC library the construction of BAC libraries even after to be 22.2 times the size of the genome. prolonged storage. This library is expected to clone a To check the quality of the BAC library, we Y-chromosome specific DNA sequence, which is preliminarily screened several genes from one-third associated with sex determination. of the BAC library (96 microtiter plates with 384 We also constructed a cosmid library using the wells). First, we screened the major frozen sperm of anti-Edwardsiella XX homozygous histocompatibility complex (MHC) genes, class Ia, clones of Japanese flounder. Edwardsiella tarda Ib, IIα and IIβ, by polymerase chain reaction (PCR) causes an infectious disease in marine fish, and assays from the super pools of BAC clone DNA or severely damages the production of aquaculture. The cell culture of the library directly. This PCR anti-Edwardsiella fish have been selected after the screening was performed by the primer sets from the challenge of the infectious bacteria and produced the cDNA sequences of MHC genes in Japanese flounder XX homozygous clone by Hyogo Prefectural (Surisapoome et al. 2004). After checking each clone Fisheries Experimental Station in 2002. We by sequencing, we found that two or three positive constructed a cosmid library from the disease- clones for each MHC gene were isolated from one- resistant fish using its frozen sperm. Analysis of the third of the BAC library (data not shown). Using the insert size of the library was performed by PFGE BAC clones, we can develop the microsatellite using the fragments of cosmid clones, which were markers related with MHC genes from their produced by NotI digestion (Figure 3A), and its size sequences, and also analyze the synteny in the MHC distribution is indicated in Figure 3B. The analysis regions between Japanese flounder and another fish indicated that the mean insert size was 39.5 kb, the species. maximum insert size was 48 kb, and the minimum insert size was 33 kb (Figure 3B). The cosmid

Okamoto 29 library consisted of 110,592 clones arrayed in 288 References microtiter plates with 384 wells. From the average Brenner, S., G. Elgar, R. Sandford, A. Macrae, B. insert size of the library, the total number of clones Venkatesh, and S. Aparicio. 1993. Characterization of the and the genomic size of Japanese flounder, the pufferfish (Fugu) genome as a compact model vertebrate analyses predicted that the cosmid library would be genome. Nature 366:265–268.

6.2 times the size of the Japanese flounder genome. Coimbra, M. R .M., O. Hasegawa, K. Kobayashi, S. This library is useful to clone a specific DNA Koretsugu, E. Ohara, and N. Okamoto. 2001. Twenty sequence, which is associated with the disease microsatellites for the Japanese flounder, Paralichthys resistance. olivaceus. Fish. Sci. 67:358–360.

Coimbra, M. R. M., K. Kobayashi, S. Koretsugu, O. Hasegawa, E. Ohara, A. Ozaki, T. Sakamoto, K. Naruse, and N. Okamoto. 2003. A genetic linkage map of the Japanese flounder, Paralichthys olivaceus. I 220:203–218.

Ciudad, J., E. Cid, A. Velasco, J. M. Lara, J. Aijón, and A. Orfao. 2002. Flow cytometry measurement of the DNA contents of G0/G1 diploid cells from three different teleost fish species. Cytometry 48:20–25.

Katagiri, T., S. Asakawa, I. Hirono, T. Aoki, and N. Shimizu. 2000. Genomic bacterial artificial chromosome library of the Japanese Flounder Paralichthys olivaceus. Mar. Biotechnol. 2:571–576.

Ojima, Y., and K. Yamamoto. 1990. Cellular DNA contents of fishes determined by flow cytometry. La

Figure 3. Analysis of insert size of cosmid clones of Anti- Kromosomo II 57:1871–1888. Edwardsiella XX clone fish. (A) NotI-digested cosmid DNAs and vector arm on PFGE. The vector size was estimated to Peterson, D. G., J. P. Tomkins, D. A. Frisch, R. A. Wing, be 7.4 kb. (B) Insert size distribution of the representative and A. H. Paterson. 2000. Construction of plant bacterial cosmid clones. The average, maximum, and minimum size of artificial chromosome (BAC) libraries: An illustrated the insert DNA are 39.5, 48, and 33 kb, respectively. guide. J. Agricul. Genomics. Online at www.ncgr.org/research/jag.

These genomic libraries are stored in the Srisapoome, P., T. Ohira, I. Hirono, and T. Aoki. 2004. National Research Institute of Aquaculture or Cloning, characterization and expression of cDNA National Research Institute of Fisheries Science of containing major histocompatibility complex class I, IIα Japan, and are available upon request for research and IIβ genes of Japanese flounder Paralichthys olivaceus. Fish. Sci. 70:264–276. purposes.

Uwa, H., and A. Iwata. 1981. Karyotype and cellular Acknowledgments DNA content of Oryzias javanicus (Oryziatidae, Pisces). The authors thank the Hyogo Prefectural Chromosome Information Service 31:24–26. Fisheries Experimental Station for providing the Japanese flounder XX clone fish. We also thank Drs. Masanori Okauchi, Takanori Kobayashi, and Kagayaki Morishima for providing technical support and Tomoyo Nozaki and Keiko Ikeyama for laboratory assistance. This research was supported by a grant from the Genomic Breeding Project by the Fisheries Research Agency of Japan.

30 Okamato Status of Pacific Bluefin Tuna (Thunnus orientalis) Seed Production in Amami Station of NCSE FRA

Hideki Nikaido, Takayuki Takebe, Kentaro Ide, and Hitoshi Imaizumi Amami Station National Center for Stock Enhancement Fisheries Research Agency 955-5, Hyo Setouchi Oshima Kagoshima 894-2414 Japan [email protected]

Nobuhiro Tezuka Notozima Station National Center for Stock Enhancement Fisheries Research Agency 15-1-1 Magarimachi, Notozima Nanao, Ishikawa, 926-0216, Japan

Syukei Masuma Miyazu Station National Center for Stock Enhancement Fisheries Research Agency 1721 Odasyukuno, Miyazu Kyoto, 626-0052, Japan

Keywords: bluefin tuna, Thunnus orientalis, seed production, stock enhancement

Abstract We have been developing seed production technology for bluefin tuna since 1995 and succeeded in rearing about 12,000 juveniles (average = 47 mm total length [TL] in 1998). However, the survival rate from hatching to the juvenile stage was typically only 0.1–0.3%. Three problems exist in seed production of bluefin tuna: viral nervous necrosis (VNN), 10 days after hatching (DAH) survival, and cannibalism occurring after 7 mm TL. The VNN was preventable by sterilizing fertilized eggs of bluefin tuna using residual oxidants in seawater and using oxidants in rearing chambers to sterilize the water. However, reasons and mechanisms of problems of the 10 DAH survival rate and cannibalism remained uncertain. We presumed that one factor affecting the 10 DAH survival rate is that bluefin larvae sink to the tank bottom during the night and subsequently die, but the cause remains unclear. We circulated the water by continuous pumping, which prevented the larvae from sinking. We examined a pump system and an aeration pipe system to produce water currents. Consequently, the 10 DAH survival rate in 2005 improved to 46%. The 10 DAH survival rate improved to greater than 60% in 2005. Survival during rearing of the early larval stage improved greatly by prevention of VNN and improved the 10 DAH survival. Using these systems, seed production technology of bluefin larvae has been steadily developing and is anticipated to progress further.

Introduction Tuna is a marine product that is cherished which serves a large market for bluefin, has promoted worldwide; its popularity is not limited to Japan. the development of stock enhancement techniques for Bluefin tuna, which has a very high market value, propagation of wild resources of bluefin tuna. grows quickly and is the largest of the tunas. The Three obstacles hinder bluefin tuna seed production: bluefin is an extremely important Japanese fishery viral nervous necrosis (VNN), the 10 days after resource. The bluefin tuna is also an international hatching (DAH) survival rate, and cannibalism fishery resource for other countries. For these occurring after the larvae reach 7 mm total length reasons, management of bluefin has been entrusted (TL). The first of those, VNN, is preventable using to international committees. Japan’s government, sterilized fertilized bluefin tuna eggs with residual Nikaido 31 oxidants in seawater (Takebe et al. 2004, Imaizumi et al. in press) and by the use of oxidant-sterilized seawater as rearing water (Tezuka et al. 2004). However, the causes and mechanisms associated with the problems of 10 DAH survival rate and cannibalism are unclear. We presumed that one cause of death before 10 DAH is that bluefin larvae sink to the tank bottom during the night and subsequently die, but the root cause remains uncertain. To address the problem, we decided to use a method of circulating water continuously using a current generator to prevent larvae from sinking. We produced currents using a pump system and an aeration pipe system. We examined the 10 DAH survival rate, the current direction and speed, and the distribution of bluefin Figure 1. Photo of pump system. The small arrows show the directions of flow of sea water and the larger arrows show the tuna larvae with respect to water depth. This paper current flow. presents the results of those studies with respect to improvement of 10 DAH survival. Aeration pipe system. The aeration pipe system Materials and Methods (Figure 2) is comprised of 1 m long, 25 mm diameter Fertilized eggs and hatching method. Takuyo air sprayer hoses. We placed 4–8 pipes on the bottom Co. Ltd. and Kinki University provided fertilized side of the wall of the rearing tank to produce an eggs of bluefin tuna used for seed production; some upward current by aeration. We did not attach air were collected from broodstock at Amami Station. stones. A polycarbonate Artemia hatching tank (200 L) was used. We sterilized fertilized eggs using residual oxidants (0.3 mg/L) in seawater before placement in the hatching tank. The eggs were hatched in oxidant-sterilized seawater.

Rearing tanks and seawater. We used octagonal concrete rearing tanks (50,000 L) at Amami Station for this study. All seawater used for rearing bluefin tuna larvae was sterilized using oxidants (0.5 mg/l) generated in the seawater by electrolysis. We used activated carbon to absorb residual oxidants.

Current generation method. We tested a method that allowed continuous rotation using two devices. One used a pump, the other used aeration pipes that are usually used for rearing fish. Figure 2. Photo of aeration the aeration system. The small arrows show air flow directions and the larger arrows show the Pump system. The pump system (Figure 1) is made current direction. of one pump put in a strainer in the center of the rearing tank and crossed plastic pipes with holes Measurement of current direction and speed. We (1.0 mm) at 10 cm intervals. Plastic pipes were divided the rearing tank bottom into 16 areas and placed on the bottom of the rearing tank, and then measured the current direction and speed at the same turned upward at 40–50° angles. The rearing point of each area. Depths of the measurement points seawater spouts were formed from holes in the were set at 5 cm, 30 cm, 1 m, and 2.5 m from the tank plastic pipe, producing an upward current. We used bottom. We used an electromagnetic current meter air stones for aeration. (ACM-300A, Alec Electronics Co. Ltd.) for

measurements. 32 Nikaido

Larval distribution. We measured the distribution density of larvae at specific points within the tank. Depths measurement points were 30 cm, 50 cm, 1 m, 1.5 m, 2 m, and 2.5 m from the surface. We used 16 mm inside diameter plastic pipes for larvae sampling. The distribution density was calculated using the number of larvae per liter of water. The survey was conducted at 1100 hours and 2100 hours on 3 DAH and 6 DAH.

Results In 2005 we reared larvae four times using the Figure 3. Comparison between survival rates of bluefin larvae pump system and twice using the aeration pipe using two ways of generating water currents. (All eggs were system (Table 1). We compared results using from the same origin.) fertilized eggs of identical origin with rearing case- 2R.

Table 1.

Figure 4. Comparison between survival rates of bluefin larvae using two ways of generating water currents in tanks. (Origin of eggs was not the same in all cases.)

Measurement of current direction and speed. We measured the direction and speed of the current in rearing water (Figure 5). Results for the pump system Survival of larvae. The 10 DAH survival rates showed a tendency for a strong upward current from using the pump system were 35.9% and 62.4%. the bottom of the rearing tank. The current speed was The 10 DAH survival rates using the aeration pipe about 4–9 cm/s upward. With the aeration pipe system were 5.5% and 6.3%. Rearing results system, the upward current speed from the bottom of showed that the pump system provided much higher the rearing tank was slower, about 2–5 cm/s, and the survival rates than the aeration pipe system (Figure current direction was irregular. 3). Particularly noteworthy is that the 62.4% result of 2R-3 became the highest result ever recorded for Larval distribution. We measured the larval rearing at Amami Station. The 10 DAH survival distribution density for every depth 3 DAH and 6 rate ranged from 20.5% to 62.4% for the pump DAH. With the pump system, the density of larvae at system in all rearing cases including when we used 1100 hours on the third DAH was similar from the fertilized eggs of different origin. This result was surface to 1.5 m depth. Larvae were present at the 2 m considerably higher than the 5.5% and 6.3% results depth, but distribution density was reduced. Larvae achieved by the aeration pipe system (Figure 4). were almost absent at the bottom of the rearing tank using the pump system. At 2100 hours, the larvae were uniformly distributed from the surface to 2.0 m

Nikaido 33 depth and were rare at the rearing tank bottom (Figure 6).

Figure 6. Density of larvae as a function of depth in the tank 3 DAH.

Figure 5. Current direction and speed in rearing tanks using two methods to generate currents. The arrows show direction of the currents.

The densities of bluefin larvae at 1100 hours and 2100 hours were almost identical from the surface to 50 cm depth. Few larvae were found at the bottom of the rearing tank (Figure 7).

In the aeration pipe system, larval density at 1100 hours on the third DAH was flat from the surface to 2.0 m depth; larvae were present at the rearing tank bottom but the density was low (Figure 6). The density of bluefin larvae at 1100 hours and 2100 Figure 7. Density of larvae as a function of depth 6 DAH. hours on the sixth DAH with the aeration pipe system was almost identical from the surface to 50 cm depth and density at the rearing tank bottom was reported, by taking natural light from the top of the low (Figure 7). rearing tank. Beginning in 2004, we examined the effects and production methods for providing a current Discussion in the rearing tanks. We succeeded in preventing Regarding prevention of the low 10 DAH bluefin larvae from sinking to the tank bottom by survival rate, Tezuka et al. (2005) examined the producing a water current with a pump system. We effects of light on the surface and current of rearing demonstrated improvement of the 10 DAH survival seawater from 1999 to 2001. Results showed the rate. The rate improved to 40% in 2003, but was about necessity of surface illumination at more than 800 10% in 2000, subsequently improving to a 60% 10 lux and the current speed of the rearing seawater at DAH survival rate in 2005. 1–2 m/s. It is possible to maintain average The purpose of generating the current is to prevent illuminance over 800 lux, as Tezuka et al. (2005) the bluefin larvae from sinking to the bottom of the

34 Nikaido rearing tanks. Sakamoto and Takashi (2005) Acknowledgments reported that bluefin tuna larvae tend to sink during Special appreciation is extended to Takuyo Co., the day, and float at night. However, during Ltd. and the Amami laboratory of Kinki University for rearing, bluefin larvae sink to the bottom of the providing valuable fertilized bluefin tuna eggs. rearing tank and die at night in most cases. This phenomenon is not explainable by gravity References fluctuation, as Sakamoto and Takashi (2005) Takebe, T., S. Masuma, N. Tezuka, H. Nikaido, and K. Ide. suggested. They reported that bluefin tuna larvae 2004. Hatching management on fertilized eggs of blue fin are prevented from sinking by swimming actively tuna thunnus orientalis disinfected by ozonated seawater. for feeding during the day; larvae sink because of NCSE Technical Report (tentative) 1:80–83. weak swimming during the night. We determined that the effect of the pump system prevented bluefin Imaizumi, H., T. Takebe, H. Nikaido, K. Ide, and S. larvae from sinking to the bottom of rearing tanks Masuma. In press. Effect of low level residual oxidants in and dying by forcibly pushing them upward using a seawater on hatching of fertilized eggs of bluefin tuna Thunnus orientalis. NCSE Technical Report (tentative). current. We prevented the VNN problem and solved the Tezuka, N., S. Masuma, T. Takebe, H. Nikaido, and K. Ide. low 10 DAH survival rate, thereby finding solutions 2004. Control of Viral Nervous Necrosis (VNN) using to two of the three salient problems inherent in ozonated seawater in seedling production of bluefin tuna, rearing bluefin larvae. As a result, the survival rate Thunnus orientalis. NCSE Technical Report (tentative). for rearing early larval stage improved 1:76–79. substantially. The cannibalism problem, however, remains unsolved and will continue as an important Tezuka, N., M. Koiso, S. Masuma, T. Takebe, H. Nikaido, subject for future study. We are reviewing the and K. Ide. 2005. Effect of water current and light on feeding protocol to determine if there is a way to survival of larval bluefin tuna, Thunnus orientalis. NCSE Technical Report (tentative), 3:41–44. mitigate growth differences of larvae and thereby control cannibalism at TL 7 mm. It appears Sakamoto, W., and T. Takashi. 2005. Gravity changes of possible to delay cannibalism by delaying the bluefin larvae. The interim research reports of 21st Century rotifer period, providing the bluefin larvae with COE Program, Center of Aquaculture Science and larval brine shrimp (Artemia nauplii), and delaying Technology for Bluefin Tuna and Other Cultivate Fish, in the start of live feeding. 2003–2004, p. 113–116. Fisheries Laboratory & Graduate Existing trials show steadily improved results School of Agriculture, Kinki University. and present the possibility of mass production of seedlings. From here, we will examine seed production techniques, other measures of mass and healthy seed production techniques, and secure genetic diversity, which will be described in future reports.

Nikaido 35

Visualization Tools to Probe Early Stage Fish Abnormalities

Susumu Uji and Tadahide Kurokawa National Research Institute of Aquaculture Research Agency, 422-1 Minami-ise, Watarai Mie 516-0193, Japan [email protected]

Keywords: abnormality, antibody, histochemical staining, visualization

Abstract The incidence of malformed fish is an important problem in the seed production of many species. The abnormal appearance of fish can be judged only when they have become large because examination or measurement of the miniscule exterior, jaws, faces, etc., of embryos and larvae is difficult. If abnormalities occur frequently, then unnecessary expenditures of human resources, money, and time must be undertaken to raise fish to sufficient size for examination. That waste would be eliminated if we could judge malformation at an early stage. To this end, we are developing rapid malformation judgment methods for fish in embryo and larval stages. Malformations are now discovered only at later growth stages, but we infer that they occur mainly during abnormal organogenesis at early stages. These malformations are detectable in early stages using visualization tools for specific organs and cells. Antibodies and histochemical staining methods are suitable for that purpose because they are effective with many fish simultaneously. Many antibodies and histochemical staining methods are useful for commercial sea fishes. For example, antibodies to acetylated tubulin, to a cell surface marker (HNK-1), and to Na K-ATPase (a5) are useful, respectively, to visualize the nervous system, neural crest cells, and kidney in fugu (Fugu rubripes), also known as puffer fish, yellowtail (Seriola quinqueradiata), and Japanese eel (Anguilla japonica). Calcein and alkaline phosphatase staining methods are also useful to visualize bones and intestines. Using these tools to probe abnormalities, we can work efficiently to solve malformation problems.

Introduction A considerable number of studies over many developmental abnormalities (Kincaid 1976, Gjerde years have examined wild and reared fish et al. 2005). Environmental factors such as abnormalities (Dawson 1964, Dawson 1966, Dawson mechanical or thermal shock, salinity, and oxygen 1971a, b). Nevertheless, numerous malformed fish depletion also have been reported to cause exist in hatchery production throughout the world developmental abnormalities (Alderdice et al. 1958, (Komada 1980, Koumoundouros et al. 1997, Garside 1959, Doroshev et al. 1974, Lee et al. 1981, Boglione et al. 2000, Cobcroft et al. 2001 and 2004, Divanach et al. 1997). Several dietary components, Fraser et al. 2005). Malformed fish are a significant such as fatty acids or vitamins, affect these problem for the aquaculture industry because abnormalities (Cahu et al. 2003). Although many malformations are often associated with growth studies exist, most examine environmental and depression and high mortality rates (Barahona- nutritional factors, especially at the juvenile stage. Fernandes 1982, Andrades et al. 1996). Their Almost none address maternal factors at early incidence engenders unnecessary expenditures of developmental stages. resources, money, and time required to raise fish in This study specifically examines this early the hatchery. Fish with malformations also depress stage—especially maternal factors—until the fish prices and lower market demand. have a first feeding. Previous studies have shown Despite their importance, the causes of that egg quality is a critical factor for survival abnormalities remain poorly understood. Genetic, (Kjørsvik et al. 1990, Furuita et al. 2003, Aegerter et environmental, and nutritional causes have been al. 2004). Most organ systems develop during the suggested as possible sources of abnormalities. For early stages of organogenesis, when organs such as example, genetic factors have been reported to cause limbs, eyes, and gills develop (Gilbert 2003).

Uji 37 Consequently, aberrant development during that Table 1. Staining method for analysis of abnormal period creates living but abnormal fish. During development in fish. organogenesis, which occurs before young fish begin to feed, the embryo develops using only egg nutrition. Most humans and domestic animals also have congenital malformations (Noden et al. 1985, Moore et al. 1998). Abnormal development is believed to be influenced by maternal factors that engender fish malformation, but no adequate technique exists to assess such problems. The embryo is extremely small and too transparent to check for abnormalities; we can obtain little information about abnormalities using only a microscope. With this consideration, we developed visualization tools to probe for early stage abnormalities. (Fugu rubripes), zebrafish, (Danio rerio), Japanese This paper represents a compilation of antibody eel, (Anguilla japonica), and yellowtail, (Seriola and histochemical staining methods we used to quinqueradiata). All these antibodies react in all visualize tissues of various fish species. We also teleosts that we examined and antigens derived from propose a concept to facilitate solving malformation nonteleost species such as mammalian species. problems. These antibodies probably react in teleosts at least.

For example, anti-HuC/HuD antibody stains Antibody Staining Methods neuronal cells in all organisms that we examined. Embryos are extremely small and are too The antibody binds specifically to antigens that are transparent to allow checking for abnormalities. For present exclusively in neuronal cells and are therefore those reasons, we can obtain little information about useful as markers of neuronal cells in tissues. Some abnormalities merely by using microscopy. To date, abnormal fish phenotypes appear to resemble human assessment of abnormalities has been conducted in neurocristopathies. Neurocristopathies are neural- most hatcheries at later stages, such as the larval or crest-cell diseases in humans. Neural crest cells juvenile stages. originate at the dorsal-most regions of the neural Visualization tools are needed to detect tube, migrate extensively, and eventually give rise to abnormalities at earlier stages such as the embryo or a remarkable variety of cell types, including neurons early larval stages. Visualization tools must also be and glial cells, cartilage, pigment cells, and readily available for many kinds of fishes. Antibody chromaffin cells. Anti-HNK-1 monoclonal antibody and histochemical staining methods meet those recognizes a carbohydrate epitope on subpopulations requirements. Using such methods, we can visualize of several cell-adhesion molecules including N-CAM their structural information. These methods are and MAG. Neural crest cells express this N-CAM. useful for many kinds of fishes. Moreover, we can Consequently, neural crest cells can be stained using deal with numerous fish simultaneously. this antibody (Suzuki et al. 2003, Ishikawa et al. The availability of antibodies and histochemical 2004). The monoclonal antibody against α- staining methods against specific cell lineages acetylated tubulin stains early axons and young facilitates the identification and localization of somata of neurons immunohistochemically (Ishikawa different organs and cells among species (Table 1). et al. 2004). Anti-a5 antibody recognizes the a- Furthermore, because of their small size, whole fish subunit of the sodium potassium ATPase (Na, K- histology can be performed to examine for ATPase, Lebovitz et al. 1989). This protein is pivotal abnormalities. to various physiological processes, including Immunohistochemical analyses were performed osmoregulation, cell volume regulation, transport of using six antibodies: anti-MF-20, anti-HuC/HuD certain amino acids and sugars, and maintenance of antibody, anti-HNK-1 antibody, anti-acetylated membrane excitability. This method enables the tubulin antibody, anti-a5 antibody, anti-myosin heavy visualization of kidney and chloride cells. chain, and clone A4 1025 antibody (Figure 1). We In addition, MF-20 antibody reacts with all performed experiments on four teleost species: fugu sarcomere myosin (Bader et al. 1982). It is possible

to view myotomes; thereby, one can speculate that

38 Uji somites give rise to muscles and the vertebral human blood tests; using it, we can readily view column, as well as the epidermal tissues. The anti- blood vessels in whole mounted samples. myosin heavy chain (clone A4 1025) antibody stains myotomes and cardiac muscle. It allows Concept to Resolve Fish Malformation visualization not only of myotomes and cardiac Problems muscle, but also eye muscles such as the superior Several important factors appear to affect the fish oblique, inferior oblique, superior rectus, and inferior in early developmental stages. These factors are rectus. All six pairs of these eye muscles are visible, classifiable as genetic, maternal, and environmental. in addition to pharyngeal muscles at later stages. Perhaps all of them cause malformation. The times at which these factors exert their respective influences are also important. First, to approach these problems, we specifically examine only maternal factors at the embryo stage. Then we hypothesize that abnormal development by maternal factors is a major cause of malformed fish. So far, however, we have only judged malformation when fish are sufficiently large to verify their shape; at this stage, it remains unclear whether fish that are being raised are normal or abnormal. As explained earlier, it is very inefficient to raise abnormal fish. Some judgment can be made earlier if the above hypothesis Figure 1. Examples of whole mount immunohistochemical is correct. Figure 2 shows a simplified scheme of an staining: a, anti-HuC/HuD antibody reacts in zebrafish early batch checking strategy with fish embryos. embryo; b, anti-myosin heavy chain, clone A4 1025 antibody reacts in fugu; c, anti-a5 antibody reacts in Japanese eel; and d, anti-acetylated tubulin antibody reacts in Japanese eel.

Histochemical Staining Methods The histochemical staining method is more useful to assess abnormalities for many kinds of fishes. Bone and cartilage are stained, respectively, using the alizarin red method and alcian blue method. These well-known histochemical staining methods are useful as visualization tools to probe fish abnormalities. Calcein is a fluorescent chromophore that binds specifically to calcium (Du et al. 2001). Figure 2. Simplified scheme of an early batch checking This method has an advantage in that it can be used strategy. to follow the development of skeletal systems in living embryos. The alkaline phosphatase staining method Because of their small size, the entire checking visualizes the intestine. Fish exhibit high levels of procedure can be accomplished within 2 days, and endogenous alkaline phosphatase, which renders the cost of this checking procedure is low. If them detectable simply by enzymatic staining development could be judged earlier, we could select (Serbedzija et al. 1999). Alkaline phosphatase exists good populations to raise, thereby eliminating the mainly in endothelial cells. Thus endothelial cells waste of resources, money, and time in raising that line the blood vessel lumen are visible, but we abnormal fish to a sufficient size to allow can see only the intestine and osteoblasts, which have examination. Environmental factors could also be high levels of endogenous alkaline phosphatase at the assessed, such as mechanical or thermal shocks, base of the tooth. That limitation notwithstanding, salinity, and oxygen depletion, in terms of their the method is sufficient because it allows inspection effects on normal organ development of fish using of the intestines. The sudan black B staining method these visualization tools. The effects of drugs and shows the blood vessels of intestines. Originally, chemicals on growth and development could be sudan black B stain was used to stain general fat in assessed in detail by examining the size and morphology of internal organs such as the brain,

Uji 39 muscle, intestines, and kidneys using these Boglione, C., F. Gagliardi, M. Scardi, and S. Cataudella. visualization tools. 2000. Skeletal descriptors and quality assessment in larvae In summary, this study revealed that many and post-larvae of wild-caught and hatchery-reared antibody and histochemical staining methods are gilthead sea bream (Sparus aurata). Aquaculture 192:1– useful for visualization of many, perhaps nearly all, 22. fishes. This paper also presented a new concept to Cahu, C., J. Z. Infante, and T. Takeuchi. 2003. resolve malformation problems. Nutritional components affecting skeletal development in fish larvae. Aquaculture 227:245–258. Acknowledgments We thank Tohru Suzuki, Hisashi Hashimoto, and Cobcroft, J. M., P. M. Pankhurst, C. Poortenaar, and B. Keiichi Mushiake for useful suggestions. The Hickman. 2004. Jaw malformation in cultured yellowtail authors are grateful to Hideki Tanaka and Yoshitomo kingfish (Seriola lalandi) larvae. N. Z. J. Mar. Freshw. Res. 38:67–71. Nagakura for providing Japanese eel and yellowtail samples, respectively. The MF-20 antibody and a5 Cobcroft, J. M., P. M. Pankhurst, J. Sadler, and P. R. Hart. antibody developed by Donald A. Fischman and 2001. Jaw development and malformation in cultured Douglas M. Fambrough, respectively, were obtained striped trumpeter Latris lineata. Aquaculture 199:267– from the Developmental Studies Hybridoma Bank 282. developed under the auspices of the National Institute of Child Health and Human Development and Dawson, C. E. 1964. A bibliography of anomalies of maintained by the University of Iowa, Department of fishes. Gulf Res. Rep. 1:308–399. Biological Sciences, Iowa City, Iowa. This study was supported in part by a grant-in-aid from the Bio- Dawson, C. E. 1966. A bibliography of anomalies of fishes: Supplement 1. Gulf Res. Rep. 2:169–176. Design Program and Development of Seed Production Technology in Japanese Eel and Japanese Dawson, C. E. 1971a. A bibliography of anomalies of Spiny Lobster Project of the Japanese Ministry of fishes: Supplement 2. Gulf Res. Rep. 3:215–239. Aquaculture, Forestry, and Fisheries. Dawson, C. E. 1971b. A bibliography of anomalies of References fishes: Supplement 3. Gulf Res. Rep. 5:35–41. Aegerter, S., and B. Jalabert. 2004. Effects of post- ovulatory oocyte ageing and temperature on egg quality Divanach, P., N. Papandroulakis, P. Anastasiadis, G. and on the occurrence of triploid fry in rainbow trout, Koumoundouros, and M. Kentouri. 1997. Effect of water Oncorhynchus mykiss. Aquaculture 231:59–71. currents on the development of skeletal deformities in sea bass (Dicentrarchus labrax) with functional swim bladders Alderdice, D. F., W. P. Wickett, and J. R. Brett. 1958. during postlarval and nursery phase. Aquaculture Some effects of temporary exposure to low dissolved 156:145–155. oxygen levels in Pacific salmon eggs. J. Fish. Res. Board Can. 15:229–249. Doroshev, S. I., and T. M. Aronovich. 1974. The effects of salinity on embryonic and larval development of Andrades, J. A., J. Becerra, and P. Fernandez-Llebrez. Eleginus navaga (Pallas), Boreogadus saida (Lepechin) 1996. Skeletal deformities in larval, juvenile, and adult and Liopsetta glacialis (Pallas). Aquaculture 4:353–362. stages of cultured gilthead sea bream (Sparus aurata). Aquaculture 141:1–11. Du, S. J., V. Frenkel, G. Kindschi, and Y. Zohar. 2001. Visualizing normal and defective bone development in Bader, D., T. Masaki, and D. A. Fischman. 1982. zebrafish embryos using the fluorescent chromophore Immunochemical analysis of myosin heavy chain during calcein. Dev. Biol. 238:239–246. avian myogenesis in vivo and in vitro. J. Cell Biol. 95:763–770. Fraser, M. R., and R. de Nys. 2005. The morphology and occurrence of jaw and operculum deformities in cultured Barahona-Fernandes, M. H. 1982. Body deformation in barramundi (Lates calcarifer) larvae. Aquaculture hatchery reared European sea bass Dicentrarchus labrax: 250:496–503. Types, prevalence, and effect on fish survival. J. Fish Biol. 21:239–249. Furuita, H., T. Yamamoto, T. Shima, N. Suzuki, and T. Takeuchi. 2003. Effect of arachidonic acid levels in broodstock diet on larval and egg quality of Japanese flounder Paralichthys olivaceus. Aquaculture 220:725– 735. 40 Uji Garside, E. T. 1959. Some effects of oxygen in relation to Lebovitz, R. M., K. Takeyasu, and D. M. Fambrough. temperature on the development of lake trout embryos. 1989. Molecular characterization and expression of the Can. J. Zool. 37:689–698. (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster. Embo. J. 8:193–202. Gilbert, S. F. 2003. Developmental Biology, 7th edition. Sinauer Associates Inc., Sunderland, Massachusetts. Lee, C. S. and B. Menu. 1981. Effects of salinity on egg development and hatching in grey mullet (Mugil Gjerde, B., M. J. R. Pante, and G. Baeverfjord. 2005. cephalus). J. Fish Biol. 19:179–188. Genetic variation for a vertebral deformity in Atlantic salmon (Salmo salar). Aquaculture 244:77–87. Moore, K., and T. V. N. Persaud. 1998. Before we are born: Essentials of embryology and birth defects. 4th Ishikawa, Y., T. Kage, N. Yamamoto, M. Yoshimoto, T. edition. WB Saunders Co., Philadelphia, Pennsylvania. Yasuda, A. Matsumoto, K. Maruyama, and H. Ito. 2004. Axonogenesis in the medaka embryonic brain. J. Comp. Noden, D. M. and A. D. Lahunta. 1985. Embryology of Neurol. 476:240–253. domestic animals: Developmental mechanisms and malformations. Williams & Wilkins Co., Baltimore, Kincaid, H. L. 1976. Inbreeding in rainbow trout (Salmo Maryland. gairdneri). J. Fish. Res. Board Can. 33:2420–2426. Serbedzija, G. N., E. Flynn, and C. E. Willett. 1999. Kjørsvik, E., A. Mangor-Jensen, and I. Holmefjord. 1990. Zebrafish angiogenesis: A new model for drug screening. Egg quality in fishes. Adv. Mar. Biol. 26:71–113. Angiogen. 3:353–359.

Komada, N. 1980. Incidence of gross malformations and Suzuki, T., Y. Haga, T. Takeuchi, S. Uji, H. Hashimoto, the vertebral anomalies of the natural and hatchery and T. Kurokawa. 2003. Differentiation of chondrocytes Plecoglossus altivelis. Copeia 1:29–35. and scleroblasts during dorsal fin skeletogenesis in flounder larvae. Dev. Growth Differ. 45:435–448. Koumoundouros, G., G. Oran, P. Divanach, S. Stefanakis, and M. Kentouri. 1997. The opercular complex deformity in intensive gilthead sea bream (Sparus aurata) larviculture: Moment of apparition and description. Aquaculture 156:165–177.

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Airlift-PolyGeyser Combination Facilitates Decentralized Water Treatment in Recirculating Marine Hatchery Systems

Ronald F. Malone and Sripavani Gudipati Department of Civil and Environmental Engineering Louisiana State University, Baton Rouge, Louisiana 70803-6405 [email protected]

Keywords: Marine hatcheries, recirculation; biofiltration, airlifts, bead filters, PolyGeyser

Abstract The nature of reproduction in fecund marine species demands that careful attention be given to the conditioning of broodstock, culture of sensitive larval stages, and production of healthy, properly sized fingerlings. Hatchery operations generally demand pristine waters and have been found to be particularly sensitive to biosecurity issues. The trend is toward the design of systems that are capable of meeting more rigorous water quality expectations in recirculating formats that display a high degree of water reuse. Traditional treatment sequences consisting of bead filters, fluidized beds, packed columns, and often pure oxygen injection systems have served the research community. These systems have also been successfully applied in the commercial production of shrimp postlarvae and ornamental fish. Increasing concerns about biosecurity are encouraging the examination of more decentralized approaches to water treatment. One such partitioned approach utilizes a pneumatic PolyGeyser floating bead filter (Aquaculture Systems Technologies LLC, New Orleans, Louisiana) in combination with airlifts to meet core recirculating needs. Use of airlifts allows the centralization of utilities without the potential of cross contamination of waters from diverse tank systems. The PolyGeyser filters are pneumatically washed in a manner that permits the recycling of backwash water, thus reducing the need for water replenishment. Sizing criteria for the bead filters, tanks, and airlift systems are provided to facilitate evaluation in the commercial sector.

Introduction It can be anticipated that future marine compatible with the emerging needs of the marine production facilities will consist of recirculating hatchery systems. hatcheries coupled with larger scale ponds, tanks, or net pen operations (Malone 2002). Recirculating Water quality targets. Malone and Pfeiffer (2006) systems are expected to continue to play an important proposed a recirculation system classification, laying role in conditioning broodstock, supporting larval a foundation for the establishment of systematic stages, and rearing fingerlings as marine aquaculture engineering criteria for treatment of recirculating production expands. These closed marine systems systems (Figure 1). They identified 17 distinct will have to be operated in a biosecure manner, recirculating classifications based on salinity, thereby assuring the health of broodstock and temperature, and trophic level. This paper fingerlings that must be held for extended periods of specifically addresses the treatment of marine time. Water quality expectations will be high, systems suitable for use as broodstock maturation and reflecting the high value placed upon healthy, fingerling growout systems in marine hatcheries. properly sized fingerlings for stocking growout Table 1 presents selected water parameters, the levels facilities. A variety of existing recirculating designs of which characterize water quality expectations for will be adapted to meet the needs of marine hatchery these hatchery systems. Establishment of these water systems. Recirculating systems will have to be quality expectations allows the formulation of designed with a relatively high hydraulic residence treatment packages that will meet the operator’s time, minimizing the opportunity for disease performance expectations. The vast majority of introduction while also minimizing discharge issues. marine production needs can be met within the This paper describes the underlying rationale for an guidelines for oligotrophic and mesotrophic systems; airlift-floating bead filter combination that is however, there is some encroachment on the eutrophic categories.

Malone 43

Conditioning of broodstock often takes months and few operations can risk loss of the time and monetary investment associated with death of breeders late in the process. Thus filtration systems are typically oversized, assuring excellent water quality. Maximum thresholds for toxic compounds (total ammonia nitrogen [TAN] and nitrite-N) are set at relatively low levels (<0.3 mg-N/L), while secondary parameters (biochemical oxygen demand [BOD] and total suspended solids [TSS]) are set at levels reflective of natural unpolluted marine systems. Broodstock are generally stocked at a low density (<15 kg/m3), a practice that, in combination with the low TAN targets, mandates high recirculation rates. Fingerling production systems focus on the production of healthy fingerlings sized for release or growout. The value of these fingerlings remains high when measured on an individual weight basis and thus justifies recirculating aquaculture systems (RAS) investment. Fingerlings are generally recognized as sensitive to water quality, so toxic water quality Figure 1. Malone and Pfeiffer (2005) identified 17 distinct parameters are set at safe but manageable levels (<0.5 types of recirculating systems. mg-N/L). Density of these systems is generally moderate (<30 kg/m3), but feeding rates can be high

(3–7%/day). Water treatment components must be Table 1. Presumed water quality targets for warm water and robust and capable of mitigating high rates of waste cold water marine hatchery systems. production. Suspended solids removal becomes critical at this stage. Solids in recirculating systems are largely organic. Uncontrolled TSS buildups induce bacterial shifts in the biofilters to the detriment of nitrifiers (Ohashi et al. 1996, Ling and Chen 2005). Many marine fingerling production systems approach densities and fingerling sizes that are more typically associated with freshwater growout. Peak TAN and nitrite levels are allowed to creep upwards (<2.0 mg N/L), a strategy that benefits from pH depression associated with rising carbon dioxide levels and the protective nature of chlorides on nitrite toxicity. Density is allowed to rise to 60 kg/m3 while the higher TAN targets lessen the importance of recirculation rates. For an example of a recirculating system, see Figure 2. Carbon dioxide removal becomes critical under these growout conditions. Rapid alkalinity consumption in combination with elevated carbon dioxide can induce pH declines that Marine broodstock maturation systems are incapacitate nitrifying bacteria. generally designed to meet the highest water quality expectations. These high standards reflect the high Current Treatment Strategies value of the eggs produced by generally fecund Floating bead filters utilize a packed bed of broodstock. Egg quality often reflects the health and floating plastic beads. They display the inherent vigor of the broodstock. ability to capture solids across a wide size spectrum.

44 Malone

microscreens for solids capture instead of a bead filter. Microscreens are often seen in combination with fluidized sand or moving bed reactors. These units are generally complemented by pure oxygen injection through a low head aerator or Speece cone with packed column carbon dioxide stripping (Summerfelt and Sharrer 2004). The unique demands of marine hatcheries are stimulating the subtle shifts in system designs (Table 2). Central is the demand of pristine waters, a clear shift towards the oligotrophic compared to the demands for high carrying capacity emphasized in the freshwater sector. Additionally, the heating and cooling needs of the systems, environmental issues associated with saltwater discharge, and, particularly, Figure 2. Example of a recirculating water treatment system. biosecurity issues are encouraging more closure of systems. Biosecurity strategies lead to designs that are more isolated and of smaller scale than Removal efficiencies decline from nearly 100% in corresponding freshwater growout systems. the 30–50 µm size range, but removal in the problematic 5–10 µm slot is high enough to assure Table 2. Water treatment configurations must be adjusted to removal with multiple passes. Thus floating bead meet the demands of marine hatchery systems. filters are able to provide for both fine and coarse solids removal irrespective of the degree of water reuse. In addition, floating bead filters are designed to encourage biofilm development and naturally provide biofiltration while they are capturing solids. Use of the floating bead filters as bioclarifiers inherently simplifies treatment trains without sacrificing water quality, laying a foundation for simplification. Malone and Beecher (2000) presented design criteria for warm water systems that have been widely applied to both freshwater and marine systems over the last decade. Their criteria support the application of pumped (circulation) floating bead filters (clarification and biofiltration) with blown air (aeration and carbon dioxide stripping) to the categories of broodstock, fingerlings, and growout It is becoming increasingly apparent that systems. This approach has been applied with many biosecurity issues will force a rethinking of variations to fingerling and growout systems. commercial marine hatchery configurations. Some engineers, however, reject the concept of Biosecurity quickly renders liberal flushing of bioclarification and take the traditional approach of systems with ambient water obsolete. Similarly, complementing the bead filter with a large biofilter. centralized water treatment where mixing of waters For example, a pumped (circulation) floating bead inherently compounds the risk of catastrophic losses filter (clarification) with a fine sand fluidized bed are unlikely to prove biosecure in a commercial (biofiltration) is a common combination. The environment. Isolation must be a cornerstone of addition of pure oxygen input through a Speece cone biosecurity strategies seeking to provide protection of (aeration) and a packed column (carbon dioxide valuable broodstock and fingerlings from internal and stripping) completes the treatment train for many external disease vectors. Partitioning of large facilities in the shrimp and ornamental fish industry. assemblies of tanks is dictated. Use of individual or A more recent variant utilizes a moving bed reactor small clusters of tanks facilitates treatment or, in the for biofiltration. In the freshwater arena a parallel set worst case, limits losses when treatment is of designs are distinguished by the use of impracticable. Partitioning also facilitates the Malone 45 manipulation of individual maturation tanks. Modern biosecure hatcheries will consist of a large number of small isolated recirculating systems. The engineering community must recognize the changing needs of the industry and focus on the development of treatment strategies appropriate for a large number of dispersed systems. To that end, this paper presents a bead filter/airlift combination that is compatible with these marine hatchery objectives. The combination addresses the five fundamental requirements of recirculation (circulation, solids capture, biofiltration, aeration, and carbon dioxide stripping) in two devices. Both devices are essentially passive, allowing the system to be operated for extended periods of time with periodic inspections. Water loss is very minimal, providing a suitable format for a biosecure operation with a very low environmental profile.

PolyGeyser filters. PolyGeyser filters (Aquaculture Systems Technologies LLC, New Orleans, Louisiana) (Figure 3) are bubble-washed floating bead filter units. They are designed to operate as a fixed bed with intermittent backwashing. The Figure 3. The pneumatic charge chamber stores air to wash distinctive difference between these units and earlier the beads then acts as a settling basin recycling backwash bead filter designs is their ability to recycle their own waters. backwash waters. The unit’s operation is pneumatic. Backwashing is implemented automatically by a slow air feed without the need for electronic timers. Thus waters captured in the charge chamber have time to there is no water loss associated with an individual settle out. A relatively clean stream of water is backwash event. Sludge can be removed from the slowly remixed with the circulating waters as the unit every few days after numerous backwashes have solids accumulate in the sludge basin. Sludge is occurred. These characteristics make the unit removed from the sludge basin usually after a great particularly suitable for marine applications. number of backwash events. Biofilms become A PolyGeyser filter has three distinctive internal abraded when a bead filter is backwashed. The components: a) the bead bed, b) the charge chamber, frequency and intensity of backwashing is a major and c) the sludge basin. The distinguishing factor that controls the nature of the biofilm, and characteristic is the charge chamber. This inverted thus, its biofiltration performance (Golz et al. 1999). compartment is designed to capture and hold a large The PolyGeyser filter is normally packed with an bubble of air. The chamber is equipped with a enhanced nitrification (EN) media. The EN media triggering device that releases the captured air when was developed to protect nitrifying bacteria during the charge chamber is filled. Then air bursts through backwashing events (Stahl et al. 2000). It is also the bead bed, rapidly mixing it as it drops. Dirty more porous than spherical beads. A bed of EN wash waters released by the bead bed rush into the media can hold about twice as much bacteria as a charge chamber, filling it as the air escapes. The spherical bead bed without displaying excessive head bead bed reforms at the top as the filter refills and loss. water filtration resumes. This backwash sequence In the mid-1990s a number of studies confirmed takes 10–15 seconds. The backwash is so fast that that a frequent but gentle backwashing strategy there is no need to interrupt the influent pump. created conditions optimum for nitrification. During the next few hours, a slow bleed of air Wimberly (1990) found the best performance of his fills the charge chamber for the next backwash event. hydraulically washed unit was eight times a day. This filling process is so slow that the backwash Sastry et al. (1998) coaxed the best performance out of his bubble-washed units at a backwash frequency of five times a day. Finally, DeLosReyes et al. 46 Malone

(1997) found his unit performed the best with a effective when they can be operated at relatively low backwash interval of only two hours. The lifts. Loyless and Malone (1998), for example, nitrification performance of filters packed with EN presented flow performance curves for a 5.05 cm media is comparable to top ranked biofilters such as airlift with a 91.4 cm submergence that showed a the moving bed reactor and fluidized sand filters. 50% decrease in flow delivery as the lift is increased The headloss displayed by EN media beds from 7.62 to 30.5 cm. subject to a high frequency washing strategy is very This issue is further complicated by gas transfer low even when subject to heavy loading. Headloss efficiencies. The gas transfer characteristics are through the bed itself is typically only a few controlled by changes in the rate of air injection (gas) centimeters. This favorable headloss profile as it relates to the water flow (liquid). It is desirable facilitates water circulation with airlift pumps. from an energy consumption perspective to operate the airlifts at low gas to liquid ratios (G:L). Air Airlift pumps. An airlift pump aerates and strips delivery rates are set to meet flow demands only. If carbon dioxide from the water as it is circulated. Of additional aeration or degassing capacity is the five basic processes that form the treatment core demanded, air is directed to the tank where both of any recirculating system, three of them can be oxygen and carbon dioxide are more efficiently substantially achieved with the simple combination transferred (Loyless and Malone 1998). of a piece of PVC pipe and an air blower. The Gudipati (2005) developed several rules of energy input that is normally reserved for the thumb for the sizing and installation of airlifts for circulating function alone is used to simultaneously water flow in recirculating applications. This work effectuate the important gas transfer functions. This concluded that airlifts should be designed to operate strategy can result in extremely simple yet robust with submergence to lift ratios in the range of four to system configurations that are very energy efficient five, keeping the G:L below two. The author also in comparison to more traditional pumped observed that lift pipes should be conservatively configurations. sized to support a water flow of 0.3 m/sec. Velocity More subtly, in contrast to centralized water recommendations for approach pipes were higher, in pumps, use of airlift pumps allows the mechanical the range of 0.6–0.9 m/sec, representing a balance of source of power to be centralized without the scour and headloss concerns. These simple rules corresponding cross contamination issues that occur facilitate the use of airlifts with a wide variety of low when waters from different tanks are mixed. This is head filtration devices. Figure 4 illustrates an airlift an important biosecurity distinction for marine configuration that has been evaluated with hatcheries. A central source of air, usually a low PolyGeyser filters ranging in size from 28 L to 1.4 head rotary vane blower, can be located in a dry m3. location away from the corrosive effects of saltwater. The air is distributed pneumatically allowing for precise localized control of the dispersed tank and filter systems without the potential for cross contamination. At the same time, centralized air pumping dramatically reduces upkeep requirements in comparison to multiple near tank centrifugal pumps. Within the context of recirculating applications, the operational performance of an airlift pump is controlled by the characteristics of the rotary vane blower that drives it. Blowers common to the industry are generally designed for air injection above a depth of 1.5 m, a depth that safely avoids gas Figure 4. Most PolyGeyser filters operate well with a screen bubble disease concerns in most applications. The placement 30.4 cm and an airlift air injection 122 cm below the tank water level. flow performance of an airlift is defined by the ratio of the lift to the submergence, with flow delivery at a fixed air input increasing dramatically as the lift The physical positioning of the PolyGeyser filter decreases. Given the constraints on depths of relative to the water level in the tank is critical. injection, recirculating airlift pumps are most cost Typically, it is necessary to place the effluent screen

Malone 47

(or pipe) 25–28 cm below the tank water level. This Table 4. These interim guidelines can be used as airlifted placement assures that there is sufficient water PolyGeysers are introduced to support recirculating aquaculture systems (metric version). pressure to push the water through the tank screens, through the tank drain lines and up though the bead bed itself under the worst case conditions. Under a variety of applications, the RAS airlifted PolyGeyser tends to operate with a cumulative head loss from tank to filter effluent in the range of 15–38 cm. This low headloss range is compatible with airlifts that have submergence depths in the range of 120–150 cm. Various engineering issues relating to the PolyGeyser filter are presented in Table 3.

Table 3. Engineering issues to be aware of in a complex airlifted PolyGeyser recirculation system.

These criteria are applicable for a temperature range of 15–30°C. Application of these criteria under peak loading outside that range should be undertaken with caution. Below a temperature of about 10°C, the overall nitrification capacity of the bioclarifier can be expected to decline. At these low temperatures, systems failure will most probably be indicated by chronic nitrite accumulation. Failure at temperatures above 30°C are most likely to be first evidenced by low dissolved oxygen levels in the rearing tank as decreasing oxygen saturation levels Airlift/PolyGeyser treatment. Table 4 provides undermine the aeration capacity of the airlifts. some interim sizing guidelines for RAS airlifted bead Supplemental, in-tank aeration may be appropriate at filters. The table presents filter feed levels and this point. approximate tank sizes for a wide distribution of Individuals installing larger units should seek PolyGeyser sizes. The feed loading indicated, professional assistance at the design and installation coupled with reasonable management of the levels to assure that the elevations for tank and filter PolyGeyser as a biofilter, should provide water are properly selected and that secondary problems quality conditions that correspond to the water such as filter flotation under high groundwater quality levels indicated in Table 1 for the different conditions are avoided (Table 4). The air delivery loading zones. Sizing for the approach pipes and the requirements of an individual unit are lift tubes associated with the airlift recirculation are straightforward. However, facilities that use multiple provided. A dual lift tube option is also sized since airlift units should seek professional advice to assure the use of two tubes facilitates energy conservation air delivery systems are properly designed. during periods of light loading. Finally the air Unexpected changes in air delivery pattern can occur delivery pressure and flow are provided to allow for as multiple units randomly backwash. Air selection of air pumps. distribution systems must be rationally laid out to 48 Malone assure that air distribution remains predictable. Summary and Conclusions Multiple airlifts serving a single PolyGeyser unit The special needs of marine hatchery systems are should return discharge water just above the tank forcing engineers to reevaluate design strategies. waterline to avoid flow reversal between competing Although current design configurations are serving airlifts. the industry well, increasing biosecurity concerns There are a number of secondary processes that dictate greater isolation and encourage high reuse can be used to complement the PolyGeyser/airlift designs that minimize exposure to external disease combination that form the core of the treatment vectors. Internally, marine hatcheries benefit from sequence (Table 5). For example, it is common the decentralization of treatment, a strategy that practice to equip broodstock conditioning tanks with reduces the potential for internal disease propagation. an ultraviolet light to suppress expression of marine The ability of the PolyGeyser floating bead filter infectious diseases or parasites. to recycle its own backwash waters facilitates the operation of recirculating systems with high Table 5. The PolyGeyser/airlift treatment core can be hydraulic retention times. This feature also permits enhanced by the addition of compatible technologies. the implementation of a high frequency backwashing

strategy that avoids headloss buildups in the bead bed. The low headlosses associated with PolyGeyser bioclarifier operation permit airlift circulation. Airlifts provide substantial aeration and degassing as they circulate, laying the foundation for a simplified treatment. An airlift PolyGeyser combination operates completely pneumatically with no moving parts. Operation is robust and requires minimal supervision, desirable features in hatchery operations with multiple isolated tank modules. Recently developed airlift sizing and established beadfilter guidelines have been merged to define interim design guidelines for airlifted PolyGeyser filters. These guidelines will be refined as commercial introductions of the airlifted PolyGeyser continue.

Acknowledgments The authors acknowledge the contributions made by commercial and other research and development units toward the refinement of this strategy. Funding for baseline studies was derived in past years from

the Louisiana and national Sea Grant College Addition of a temperature control loop facilitates programs. Commercial development was fostered by temperature manipulations used for condition in a Department of Commerce grant and several U.S. broodstock. Moving bed reactors and rotating Department of Agriculture Small Business Innovative biological filters are examples of two biological Research Grants conducted by Aquaculture Systems filters that can be used in an airlift recirculating Technologies Inc., New Orleans, Louisiana, under system. A number of foam fractionator designs are the direction of Douglas Drennan. Burt Nichols at air driven and compatible with the pneumatic Water Garden Gems Inc., Marion, Texas, has been operation of the airlifted PolyGeyser. Foam active through donations of equipment for fractionators are most frequently installed on heavily commercial demonstrations. loaded fingerling systems, or on broodstock systems An early commercial demonstration (circa 1995) that are operated with high hydraulic residence times of the airlift technology with a paddle-washer was (>5 days). Denitrification also becomes necessary conducted by Steve Abernathy of Tiltech Inc., when water reuse extends hydraulic residence times. Robert, Louisiana. That demonstration, which continues today, formed the driving force for the continued research efforts. Waddell Mariculture Center in South Carolina formed an early test base

Malone 49 for small-scale airlift evaluations, as did the Centro Malone, R. F., and L. E. Beecher. 2000. Use of floating de Investigacion Cientifica y de Educacion Superior bead filters to recondition recirculating waters in de Ensenada (CICESE) in Baja, Mexico. Hubbs-Sea warmwater aquaculture production systems. Aquacult. World Research Institute in San Diego, California, Eng. 22:57–73. has an ongoing evaluation of a 25 cubic foot airlift Malone, R. F. 2002. Engineering for a responsible unit. aquaculture with focus on growout facilities for Most notably, Mote Aquaculture Park, Sarasota, commodity foodfish. In R. L. Creswell and R. Flos (eds.), Florida, is conducting the largest demonstration Perspectives on responsible aquaculture for the new 3 3 project consisting currently of four 1.5 m (50 ft ) millennium, p. 94–111. World Aquaculture Society, Baton PolyGeyser units on freshwater sturgeon, while Rouge, Louisiana, and European Aquaculture Society, evaluating several PolyGeyser Moving Bed Oostende, Belgium. combinations on their marine broodstock systems. These pioneers identified the limitations of our Malone, R. F., and T. J. Pfeiffer. 2006. Design and knowledge as this emerging technology was selection of biological filters for freshwater and marine introduced. applications. Aquacult. Eng. 34(3):389-402.

Ohashi, A., D. G. Viraj de Silva, B. Mobarry, J. A. References Manem, D. A. Stahl, and B. E. Rittmann. 1996. Influence DeLosReyes Jr., A. A., L. E. Beecher, and R.F. Malone. of substrate C/N ratio on the structure of multi-species 1997. Design rationale for an air-driven recirculating biofilms consisting of nitrifiers and heterotrophs. Water system employing a bubble-washed bead filter. In M. B. Sci. Technol. 32(8):75–84. Timmons and T. Losordo (eds.), Advances in aquacultural engineering: Aquacultural Engineering Society Sastry, B. N., A A. DeLosReyes Jr., K. A. Rusch, and R. Proceedings III, ISTA IV, Orlando, FL, 9–12 November F. Malone. 1999. Nitrification performance of a bubble- 1997, p. 287–294. NRAES, New York. washed bead filter for combined solids removal and biological filtration in a recirculating aquaculture system. Golz, W. J., K. A. Rusch, and R. F. Malone. 1999. Aquacult. Eng. 19:105–117. Modeling the major limitations on nitrification in floating- bead filters. Aquacult. Eng. 20:43–61. Summerfelt, S. T., and M. Sharrer. 2004. Design implication of carbon dioxide production within biofilters Gudipati, S. 2005. Distributed airlift systems design with contained in recirculating salmonid culture systems. application to recirculating soft shell crawfish shedding Aquacult. Eng. 32:171–182. systems. Master’s thesis. Louisiana State University, Baton Rouge. Wimberly, D. M. 1990. Development and evaluation of a low-density media biofiltration unit for use in recirculating Ling, J., and S. Chen. 2005. Impact of organic carbon on fish culture systems. Master’s thesis. Louisiana State nitrification performance of different biofilters. Aquacult. University, Baton Rouge. Eng. 33:150–62.

Loyless, J. C., and R. F. Malone. 1998. Evaluation of airlift pump capabilities for water delivery, aeration, and degasification for application to recirculating aquaculture systems. Aquacult. Eng. 18:117–133.

50 Malone Preparation of Marine Silage and its Potential for Industrial Use

Motoharu Uchida National Research Institute of Fisheries and Environment of Inland Sea 2-17-5 Ohno, Hatsukaichi Hiroshima, Japan 739-0452 [email protected]

Keywords: hatchery diet, lactic acid fermentation, marine silage, seaweed

Abstract Marine silage (MS) as used here is a dietary material prepared from algae by a fermentation process. The objective of this paper is to report on the method used to prepare MS from seaweeds and discuss its potential for industrial use. The fermentation of seaweeds can be performed by the enzymatic saccharification by cellulase, followed by fermentation using lactic acid bacteria and/or yeast. The method can be applied to any kind of seaweed containing cellulose. Red sea bream that were challenged with iridovirus and fed a diet containing MS at 10% (w/w) of the diet showed a significantly improved survival percentage compared with those fed a control diet. The use of MS in the diet may allow for the production of cultured fish that are free of drugs. In the case of Asian kelp (Undaria pinnatifida), the fronds are fragile and easily decomposed during the saccharification process. The particle size of the product obtained (single cell detritus-marine silage, or SCD-MS) is ca. 5~10 µm in diameter, similar to a microalgae diet. The mass culture of the SCD-MS can be performed in a plastic tank, with concentrations reaching ca. 107–108 cells/mL. Supplemental aeration, temperature control, and light control are not necessary. Feeding trials with pearl oyster (Pinctada fucata martensii) spat demonstrated the dietary value of the SCD-MS to bivalves. The SCD-MS diet may replace a part of microalgae diets in the future. The development of the SCD-MS diet also suggests a new style of aquaculture. We are attempting to link the production of nuisance algae such as Ulva spp. to fish production utilizing the marine silage system. We believe that aquaculture based on algal fermented materials is ecofriendly.

Introduction Fermentation has a long history and plays an applied to any type of seaweed that contains cellulose important role in agriculture and the livestock (Table 1, Uchida and Murata 2004). industry for preparing compost and silage. However, fermentation is not extensively used to feed fish except for some limited applications in ingredient manufacturing. Particularly lacking has been the approach of fermenting algae, which are plentiful in marine ecosystems. In fact, there have been no fermented food products made from marine algae (Figure 1). Recently, the author developed a method to perform lactic acid fermentation on seaweeds (Uchida and Murata 2002). The method contains two elements (Figure 2a). The first is the enzymatic decomposition and saccharification of seaweeds. The second is lactic acid fermentation using microorganisms. These two processes can be performed simultaneously by adding polysaccharide- decomposing enzymes; usually cellulase, and starter microorganisms; usually lactic acid bacteria (LAB) or yeast to initiate the process. This method can be Figure 1. Categories of fermented food products.

Uchida 51 Table 1. Results of the fermentation test for various kinds of seaweed with the addition of cellulase and the microbial starter mixture (Uchida and Murata 2002).

Figure 2. Fermentation process of seaweed. Lactic acid fermentation (a) and lactic acid fermentation with single cell detritus (SCD-MS) products (b).

Materials and Methods Preparation of SCD-MS for determining appropriate culture conditions. To determine the appropriate culture conditions for SCD-MS, sterile 500 mL polycarbonate bottles with screw caps (Nalgene) were used for a small scale fermentation trial. A commercial product of dried particles of U. pinnatifida (Wakamidori <74 µm, Riken Co.) was dispensed in the amount of 10.0 g each into bottles containing 180 mL of autoclaved 3.5% w/v NaCl solution and cellulase (R-10, Yakult Pharmaceutical The author is promoting the use of this fermented Ind. Co.) (Table 1) and mixed well. The culture material from seaweed as a fish dietary ingredient bottles were capped and incubated at 20°C with with the proposed name marine silage (MS). shaking several times a day to improve dispersion. Furthermore, in the case of Asian kelp (Undaria Initially, the preparation of SCD in cultures without pinnatifida), the observation was made that the frond the addition of a microbial mixture was conducted tissue can be decomposed to one-cell units by under various reaction temperatures and cellulase cellulase during fermentation (Figure 2b). As a concentrations. The number of detrital particles of result, large numbers of small detrital particles (ca. Undaria obtained was counted using a Coulter 5–10 µm in diameter) are produced. The author Multisizer (Coulter Electronics Ltd.) with a 140 µm termed these particles single cell detritus-marine orifice. The detrital particles in the fractions of 5.8– silage (SCD-MS) and studied the optimal culture 11.5 µm in diameter were tentatively regarded as conditions for preparing the material (Uchida and SCD-MS products. The weight distribution was Murata 2002). calculated from the distribution of the detrital This paper reports on the suitable culture particles on the assumption that the detrital particles conditions for performing lactic acid fermentation of were of a spherical form with a specific gravity of seaweed and for preparing SCD-MS with high 1.0. efficiency. Furthermore, to demonstrate the potential For testing the effect of the use of microbial of the MS as fish dietary ingredient, a feeding trial elements as a starter, cultures containing different was conducted with red sea bream. Since the size of levels of cellulase were incubated with an inoculation the SCD-MS is analogous to microalgae diets such as of the microbial mixture. The microbial mixture was Chaetoceros calcitrans and Pavlova lutheri, a composed of one type of LAB Lactobacillus brevis feeding trial with young pearl oysters (Pinctada strain B5201 (FRA 000033) and two types of yeast: fucata martensii) was also conducted to demonstrate Debaryomyces hansenii strain Y5201 (FERM BP- the dietary value of the SCD-MS as a replacement for 7302) and Candida sp. strain Y5206 (FERM BP- microalgae diets (Uchida et al. 2004a). 7303). The bacterium and yeast cells were

52 Uchida preincubated in MRS broth (Merck) and YM broth Rearing trial for red sea bream challenged with (Difco), respectively, collected by centrifuge, washed iridovirus. The Ecklonia-MS was prepared by twice, and suspended in sterile 0.85% NaCl solution fermenting the Ecklonia maxima (a dried product at a concentration of OD 660 nm = 1.0. The imported from South Africa) at 10°C for one month suspension mixture of 2 mL of L. brevis (containing after the addition of 0.15% (as a final conc.) cellulase 1.0 × 109 cells) and 1 mL each of D. hansenii (5.3 × 3S (Yakult Pharmaceutical Ind. Co.), 0.035% 105 cells) and Candida sp. (1.2 × 107 cells) was used pectinase G (Amano Enzyme Co.), and 8 × 106 for inoculation of the microbial mixture. All cultures CFU/mL L. plantarum (TOA Pharmaceuticals Co.). were prepared in duplicate and the average values of The Ecklonia-MS diet was prepared by adding the data were determined. Ecklonia-MS to a control diet at 10% w/w. The composition of the control diet was 40 g brown meal, Mini preparation of SCD-MS. A sterile 50 mL 20 g white meal, 10 g northern krill meal, 7 g sardine volume polypropylene centrifuge tube provided with oil, 8 g dextrin, 10 g wheat gluten, 3 g guar gum, 1 g a screw cap (Iwaki) was used to prepare the vitamin mixture, 0.1 g mineral mixture, 0.66 g fermentation cultures. To prepare NaCl-added CaCO3, 0.23 g CaPO4, and 0.01 g Hospitane. For the cultures (No. 1a–14a), 2.0 g of the Undaria product rearing trial, red sea bream (mean ±SD of body were mixed with 40 mL of autoclaved 3.5% NaCl weight = 32.9 g ±3.2 g) were challenged with 50 µL solution, 40 mg of cellulase 12S (Yakult each of iridovirus stock suspension (TCID50 = Pharmaceutical), and 0.4 mL of bacterial cell 105.8/mL). Then 50 individuals were held in each of suspension and mixed. The bacterial cell suspension three 200 L tanks and reared at 25°C. The rearing was prepared for the 14 LAB strains: L. brevis FRA water was changed at the rate of 1L/min. The fish 000033, L. brevis IAM 12005, L. plantarum ATCC were fed to satiation twice a day. 14917T, L. plantarum IAM 12477T, L. casei IFO 15883T, L. casei FRA 000035, L. rhamnosus IAM Rearing trial for young Japanese pearl oysters. 1118T, L. zeae IAM 12473T, L. acidophilus IFO For the preparation of MS for the rearing test, 20 g of 13951T, L. kefir NRIC 1693T, L. fermentum ATCC the Undaria product was dispensed into 500 mL 14931T, L. delbrueckii bulgaricus ATCC 11842T, polycarbonate bottles containing 360 mL of 3.5% Streptococcus thermophilus NCFB 2392, and NaCl solution and 2 g cellulase R-10 and mixed well. Leuconostoc mesenteroides IAM 13004T. The strains The cultures were inoculated with 2 mL of the cell were precultured with MRS medium, collected by suspensions of the previously described three types of centrifuge, resuspended to make a concentration of microorganisms and incubated for 8 days at 20°C OD 660 nm = 1.0 (containing 7.3 × 107–1.1 × 109 (SCD-MS2). A culture that was prepared without CFU/mL), and used. To prepare non-NaCl cultures addition of the microorganisms and incubation was (No. 1b–14b), autoclaved distilled water was used used as a control diet (C-MS2). The SCD-MS2 and instead of 3.5% NaCl solution. Cultures without C-MS2 were preserved at 5°C during the rearing test LAB inoculation were prepared as No LAB controls and filtered through 6.3 µm nylon mesh just before (No. 15a and 15b). The triplicate cultures were use. Early stationary phase cells of Chaetoceros incubated at 20°C for 11 days mixing several times calcitrans were prepared by culture at 20°C (14 h:10 by hands every day to help dispersion. h light:dark cycle) in a modified Erd-Schreiber medium. For the rearing trial, 15 young Japanese Mass preparation of SCD-MS. Sterile 10 L pearl oysters (mean of hinge length = 4.47–4.95 mm) polycarbonate bottles with screw caps (Nalgene) were held in a 5 L tank and reared for 12 days. The were used for the preparation of MS. One kg of U. rearing water was supplied with air and kept at a pinnatifida Wakamidori was dispensed into bottles temperature of 27°C and a salinity of 32–33 ppt. containing 9 L of autoclaved 3.3% NaCl solution and Oysters were fed once a day and the rearing water 10 g cellulase R-10 and mixed well. The cultures was changed every day with fresh, sand-filtered were inoculated with the microbial mixture at seawater. The control groups were an unfed group, a quantity of 5.0 × 1010 CFU (L. brevis), 5.5 × 107 CFU Chaetoceros group (3 × 104 cells/mL/day), and a 1/10 (D. hansenii) and 1.2 × 108 CFU (Candida sp.). Each Chaetoceros group (3 × 103 cells/mL/day). The test culture bottle was capped, incubated at 20°C and groups were those fed the SCD-MS at 2 × 104 mixed several times a day to improve dispersion for cells/mL/day (SCD-MS2) and 5 × 104 cells/mL/day the first eight days. The culture bottles were then (×2.5 SCD-MS2). The combined feeding was tested preserved at 20°C for 18 months. by feeding the MS2 at 2 × 104 cells/mL/day with Chaetoceros at 3 × 103 cells/mL/day (SCD-MS2 + Uchida 53 1/10 Chaetoceros). The test groups using the C-MS2 odors and were judged to have rotted. In contrast, the diet were presented with the same feeding conditions culture media containing cellulase at 0.25–5.0% had as the SCD-MS2 groups. Shell length of the spat’s ester-like preferable odors and were judged to be hinge lines (hinge length) were measured and used fermented. for calculating growth rates. The survival rate of the For the cultures without cellulase, microbial flora spat was obtained by counting the number of living after 5 days of culture varied greatly, implying individuals at the end of the rearing trials. unstable culture conditions from the viewpoint of microbial composition. One culture contained Results and Discussion predominantly lactic acid bacteria (80.5% of Suitable culture conditions for preparing the heterotrophic microorganisms) while the other SCD-MS. The relationship between the culture contained predominantly marine heterotrophic temperature and the number of SCD-MS products microorganisms (92.9%). For the fermented cultures, 8 was studied with a culture containing 3.5% NaCl and lactic acid bacteria were predominant at a 10 1.0% cellulase. The volume proportion of SCD-MS CFU/mL level and the ratio of lactic acid bacteria to increased gradually and reached a maximum value in heterotrophic microorganisms was in the range of the range of 60.1–69.0% on the fourteenth day 8.5–11%, while the proportions of marine (Figure 3). The production efficiency of SCD-MS heterotrophic against heterotrophic microorganisms was maximum at 2°C and almost constant in the were in the range of 3.6–8.0%. It was observed that range of 20–50°C based on the profile of Figure 3. the addition of 0.25% cellulase to the culture media The production efficiency of SCD-MS was maximum was enough to obtain SCD-MS without spoilage if at 2°C and almost constant in the range of 20–50°C the starter microorganisms were added (Table 2). In based on the profile of Figure 3. contrast, the addition of cellulase more than 0.5% was necessary to obtain SCD-MS without spoilage if the starter microorganisms were not added.

Figure 4. Relationship between cellulose concentration and production efficiency of SCD-MS.

Figure 3. Relationship between culture temperature and production efficiency of SCD-MS. Table 2. Cellulase concentration necessary for successful fermentation.

Figure 4 shows a profile of the relationship between cellulase concentration and the number of SCD-MS in Undaria cultures prepared in the presence of starter microbes. The number of SCD- MS increased from 2.5 × 107/mL (18.4% based on 7 volume proportion) at the start to 3.5–5.9 × 10 /mL (63.0–69.1% based on volume proportion) at 6 days of incubation and the maximum SCD-MS products were obtained with a cellulase concentration at 0.5% The starter microorganisms were first obtained (59.1% based on volume proportion). Culture media from a naturally fermented material of Ulva spp. without the addition of cellulase had unacceptable Three species of microorganisms, namely L. brevis,

54 Uchida D. hansenii and Candida sp., were isolated as predominant microorganisms in the fermented Ulva. We observed that the addition of the individually cultured cell suspensions of these microorganisms as a starter for fermentation of seaweed was effective in the production of SCD-MS (Uchida and Murata 2004). However, we also found that lactic acid bacteria alone can be used as a starter (Uchida et al. 2004b). Results of fermentation with NaCl-added cultures are shown in Figure 5a. The predominance of the inoculated species in the cultures was 100% for the cases of L. casei IFO 15883T and L. casei FRA 000035; 96.7% for the cases of L. brevis FRA 000033, L. brevis IAM 12005, and L. plantarum IAM 12477T; 93.3% for the case of L. plantarum ATCC Figure 5. Microbial compositions after fermentation of 14917T; 90.0% for the case of L. rhamnosus; 13.3% Undaria with inoculation of different starters in NaCl-added cultures (a) and cultures lacking NaCl (b). Starter strains No. for the case of L. fermentum; and less than 3.3% for 1-14 are: L. brevis FRA 000033, L. brevis IAM 12005, L. the cases of L. zeae, L. acidophilus, L. kefir, L. plantarum ATCC 14917T, L. plantarum IAM 12477T, L. delbrueckii bulgaricus, S. thermophilus, and casei IFO 15883T, L. casei FRA 000035, L. rhamnosus IAM Leuconostoc mesenteroides. The LAB became 1118T, L. zeae IAM 12473T, L. acidophilus IFO 13951T, L. kefir NRIC 1693T, L.fermentum ATCC 14931T, L. predominant (>90.0%) for 13 cases out of 14, while delbrueckii subsp. bulgaricus ATCC 11842T, Streptococcus the inoculated species became predominant for 7 thermophilus NCFB 2392, and Leuconostoc mesenteroides cases out of 14. IAM 13004T. Culture 15a and 15b are control cultures Results of fermentation with NaCl-less cultures prepared without starter inoculation. are shown in Figure 5b. The predominance of the starter species was 100% for the cases of L. casei IFO 15883T, L. casei FRA 000035, and L. rhamnosus; the results of a feeding trial conducted on young pearl 96.7% for the cases of L. brevis FRA 000033 and L. oysters. The feeding of Chaetoceros calcitrans at 3 plantarum ATCC 14917T; 90.0% for the case of L. ×104 cells/mL/day resulted in a shell growth of 168 plantarum IAM 12477T; 63.0% for the case of L. µm/day and survival of 66.7%, while unfed controls brevis IAM 12005; 3.3–20.0% for the cases of L. showed negative shell growth (–10 µm/day) and zeae, L. kefir, L. fermentum, and Leuconoscoc 53.3% survival. Feeding with a SCD-MS prepared 4 Mesenteroides; and less than 3.3% for the cases of L. from U. pinnatifida at 2 × 10 cells/mL/day resulted acidophilus, L. delbrueckii bulgaricus, and in a shell growth of 23 µm/day and survival of Streptococcus Thermophilus. The LAB became 53.3%. When the SCD-MS was supplied with C. 3 predominant (>90.0%) for 10 cases out of 14, while calcitrans at 3 × 10 cells/mL/day, shell growth was the inoculated species became predominant for the 6 130 µm/day and survival was 80%. This study cases out of 14. L. brevis, L. plantarum, L. casei, and demonstrates the dietary value of SCD-MS to bivalve L. rhamnosus predominated in the MS cultures. mollusks and shows that SCD-MS can partially replace microalgal diets in hatcheries. Promotion of survival rate of fish against viral disease. Red sea bream challenged with iridovirus Table 3. Results of feeding trials of SCD-MS2 and C-MS2 with young pearl oyster (Uchida et al. 2004a). and fed a diet containing the MS showed significantly better survival rate (86.5% ±1.6%) compared with those fed the control diet (73.1% ±6.3%). The use of MS diet may help increase the supply of fish that are produced without the use of drugs, which is an important consideration for consumers today.

SCD-MS as a replacement hatchery diet for microalgae diet. Since the size of the SCD-MS is about 5–10 µm in diameter, it has a potential to replace microalgal diets in hatcheries. Table 3 shows Uchida 55 New Aquaculture Style Based on Marine References Silage Uchida, M., and M. Murata. 2002. Fermentative The development of the MS preparation preparation of single cell detritus from seaweed, Undaria technology and its widespread use may change the pinnatifida, suitable as a replacement hatchery diet for way aquatic animals are fed in the future. Figure 6 unicellular algae. Aquaculture 207:345–357. presents the concept of aquaculture based on a Uchida, M., and M. Murata. 2004. Isolation of a lactic sustainable and ecofriendly marine silage system. acid bacterium and yeast consortium from a fermented There has been an increase in nuisance blooms of material of Ulva spp. (Chlorophyta). J. Appl. Microbiol. Ulva spp. and other green algae in the eutrophic 97:1297–1310. coastal waters of many countries. The blooms are believed to be the result of excessive nutrients such Uchida, M., K. Numaguchi, and M. Murata. 2004a. Mass as nitrogen and phosphate in coastal waters. preparation of marine silage from Undaria pinnatifida and Therefore, collection and utilization of algal fronds its dietary effect for young pearl oyster. Fish. Sci. 70:456– could contribute to decreases in coastal water nutrient 462. levels. Another likely advantage in the use of SCD- Uchida, M., H. Amakasu, Y. Satoh, and M. Murata. MS diets is that the microorganisms present in the 2004b. Combinations of lactic acid bacteria and yeast SCD-MS media may have probiotic or prebiotic suitable for preparation of marine silage. Fish. Sci. effects on the animals being reared. We are currently 70:508–518. working on that potential for SCD-MS.

Figure 6. Concept of eco-friendly aquaculture based on a marine silage diet.

56 Uchida Techniques for Live Capture of Deepwater Fishes with Special Emphasis on the Design and Application of a Low-cost Hyperbaric Chamber

Jeffrey E. Smiley and Mark A. Drawbridge Hubbs-SeaWorld Research Institute 2595 Ingraham Street San Diego, CA 92109 [email protected]

Keywords: hyperbaric chamber, rockfish, gas bladder

Abstract Following rapid depressurization from depth, physoclistous fish suffer from overinflation of their gas bladder. The decreased pressure also causes bubbles to form in the circulatory system and causes trauma to internal tissues resulting in hemorrhage, swelling, and death. Our research has shown that Cowcod (Sebastes levis) can only survive a few hours after rapid depressurization from depths as shallow as 90 m. To overcome this problem, we developed a relatively simple, low cost hyperbaric chamber to aid in the collection of adult rockfish to hold as broodstock. This system was designed to quickly recompress fish once brought to the surface on hook and line, and allow for decompression over a period of days. The hyperbaric chamber is capable of continuous stable operation at <10.2 atmospheres and can accommodate fish up to 91.4 cm in length and 26.8 cm in diameter. Pressure in the chamber is maintained by a Goulds booster pump that delivers continuous pressure and supplies flows of 3.8–7.6 L/min to as many as four chambers. The hyperbaric chamber operated very effectively and allowed us to successfully decompress 14 cowcod and two vermillion rockfish (Sebastes miniatus) from capture depths of 90.2-146.3 m.

Introduction When fish are captured at depth, rapid chambers have been developed. These hyperbaric depressurization can cause serious injuries, including chambers are typically designed to trap fish at depth gas bubbles in the blood vessels, gills, skin, and and return them to the surface under pressure brain; exophthalmia; internal and external (Jannasch et al. 1973, Yayanos 1978, Macdonald and hemorrhage; everted stomachs; disoriented Gilchrist 1978, Wilson and Smith 1985, Koyama et swimming; altered behavior; and death (Gotshall al. 2002). 1964, Beyer et al. 1976, Feathers and Knable 1983, Hyperbaric fish traps have made it possible to Rogers et al. 1986, Gitschlag and Renaud 1994). collect specimens from the deep sea (600–5,700 m) Specialized collection techniques are needed to but they are very costly to design and build, thereby minimize barotrauma among deepwater fishes that creating the need for a low cost solution, especially are collected live for public display or breeding for fish at shallower depths. To meet this need, we programs. Collection techniques will vary based on developed a portable hyperbaric chamber that is the depth of capture and sensitivity of the target capable of recompressing fish brought to the surface, species. then maintaining them at pressures up to 10.2 Techniques used to collect fish from depth atmospheres for weeks. We targeted several species typically include cages, trawls, and hook and line of rockfish (Sebastes sp.) with the goal of holding the fishing. Without special handling, fish collected fish for breeding purposes. using these methods are typically either moribund or dead. Special handling techniques include gradual Hyperbaric Chamber Design and decompression and gas bladder deflation (Gotshall Construction 1964, Lee 1992, Keniry 1996). Gas bladder deflation Design criteria. The hyperbaric chamber described has enabled collection of some specimens from the in this paper was designed based on the following sea, but mortality is highly variable and increases criteria: with depth. To increase survivability, more time 1. Portability to allow manageable movement of must be taken to decompress fish. In order to fish from the field to the laboratory under accomplish this, specialized pressure-controlled controlled conditions. Smiley 57 2. Low cost using standardized parts that are common and relatively inexpensive. 3. Pressure stabilization at 150 psi to allow complete recompression of fish caught at depths of 102.7 m. 4. Temperature control to maintain constant conditions from the point of capture thereby reducing stress on the fish. 5. Visual monitoring to ascertain fish health and make decisions for safe decompression Figure 1. Portable primary manifold and booster pump schedules. attached to a hand truck for ease of transport. 6. Water quality monitoring to ensure stable seawater temperature and adequate dissolved oxygen concentrations. four chambers that allowed individual pressure and flow control, safety, and bypass loops. Water from Designing the rest of the apparatus to meet our the primary manifold was diverted through a check portability goals required a multipart decompression valve and a needle valve designed to control the system that could be broken apart in manageable amount of water delivered to the chamber. Water sections. These sections included a primary and could enter or bypass the chamber through a 1.9 cm secondary manifold and chamber. The primary ball valve. This bypass allowed operation of the manifold was responsible for maintaining stable secondary manifold without seawater flowing pressures for the secondary manifolds, the secondary through the chamber. After seawater was routed manifolds were responsible for maintaining stable through the chamber or bypass, it was pushed pressures and flows in the chambers, and the through a t-strainer before being routed through a 0.6 chambers were responsible for specimen containment cm pressure relief valve. Downstream of the pressure and pressure isolation for greater than 30 minutes; the relief valve, a 1.0 cm normally closed solenoid valve time allowed for transport from the boat back to the was installed that operated as a safety valve when lab. system power failed. Seawater that had passed the solenoid valve was routed through a rotary flow Principle component: Primary manifold. Pressure indicator and two 0.6 cm labcock valves used to in the primary manifold was created by a 3.0 hp divert outgoing flow to a cylindrical reservoir where Goulds booster pump (25GBS3014P4) that delivered water quality measurements could be taken. Two 94.6 L/min of seawater to the apparatus. Pressure in other 1.3 cm ball valves were also used to bypass the the system was controlled by a 2.54 cm pressure solenoid and pressure relief valves in the event of relief valve that vented excess seawater back to the component failure or operation at ambient pressure sump and created stable backpressure in the system. (Figure 2). A portion of the backpressured seawater was diverted before the pressure relief valve and served as the source for the secondary manifolds. Four 1.9 cm ball valves operating in parallel from the primary manifold controlled the flow of seawater to four secondary manifolds through sections of 1.3 cm flexible hose. The hose had a maximum operating pressure of 20.4 atmospheres and ranged in length from 4.6 to 6.1 m. Pressure in the primary manifold was monitored on a 10.1 cm face gauge up to 13.6 atmospheres. Two 5.1 cm ball valves were also installed on the primary manifold and opened during startup procedures to reduce initial water-hammering. For portability the primary manifold and pump was bolted to a hand truck (Figure 1). Figure 2. Schematic of secondary manifold and chamber Principle component: Secondary manifold. A control valves. secondary manifold was constructed for each of the 58 Smiley Principle component: Chamber. The hyperbaric Pressurization sequence. Prior to receiving a fish, chamber was constructed from 30.5 cm schedule 80 each chamber was set on its end (open side up) and PVC pipe with an internal diameter of 28.6 cm. It filled with seawater (Figure 4). Water pressure was was cut 91.4 cm in length and had two 30.5 cm regulated and stabilized in the primary manifold at flanges glued to each end, increasing internal length 10.9 atmospheres and in the chamber bypass line of to 95.3 cm. On one end a 5.1 cm-thick acrylic the secondary manifold at 8.8 atmospheres. Once a window and gasket were bolted to the flange with 12 fish was caught and brought to the surface, it was stainless steel bolts. Two 1.3 cm bulkhead fittings placed inside the chamber head down. A blind flange were installed in the side of the cylinder 50.8 cm and gasket assembly was then attached to the top of apart. A 1.3 cm ball valve was connected to each of the chamber with 12 stainless steel bolts. The bolts the bulkhead fitting with two 5.1 cm face pressure were torqued in a star pattern to 13.1 m/kg. Once gauges located on opposite sides of the valves sealed, the valves isolating the chamber were opened, coming in and out of the chamber. A wood cradle which equalized pressure from the secondary was designed for the chamber allowing it to be manifold. This began the recompression sequence. strapped to a 600 lb capacity hand truck (Figure 3). Once at the maximum pressure, the valve bypassing The secondary manifold was strapped beneath the the chamber on the secondary manifold was closed chamber. Connections from the chamber to the forcing seawater to flow through the chamber. secondary manifold were made with a 1.3 cm flexible hose rated at 20.4 atmospheres. A PVC blind flange and gasket were attached to the open end of the cylinder to complete the chamber. Pressure in the secondary manifold and chambers reached a maximum of 9.5 atmospheres.

Figure 4. Hyperbaric chamber set on end prior to fish insertion.

Monitoring environmental conditions and Figure 3. Hyperbaric chamber and secondary manifold (not in view) attached to a hand truck for ease of operation and chamber water quality. Subsurface sea conditions portability. were monitored using a Hydrolab DataSonde. The data were used to identify target water quality parameters for chamber operations and broodstock Hyperbaric Apparatus Operation maintenance. The Sonde was factory fit with depth Seawater supply. Seawater supply to the apparatus (±0.03 m), dissolved oxygen (±0.01 mg/L), depended on its location—either in the field or the temperature (±0.01°C), pH (±0.01 unit), salinity laboratory. During field operation, the primary (±0.01 ppt), and PAR (±1). Measurements were manifold was supplied with chilled water from the recorded every 30 seconds as the probe was lowered bait well onboard the vessel using a 0.5 hp Flotec to depth at targeted fishing areas. A Reefnet Sensus submersible sump pump. In the laboratory, seawater Pro depth recorder was used to record changes in was pumped from Mission Bay (San Diego, pressure as the fish were being caught by hook and California), filtered using rapid sand filters, and line, recompressed, and decompressed in the chilled in a recirculating sump. The temperature chamber. The depth recorder was programmed to log regulation systems at both locations were computer data every 10 seconds when attached to the controlled, resulting in consistent supply fisherman’s line, and every 30 seconds when placed temperatures.

Smiley 59 in the chamber. The Reefnet logger measured depth Field Trials to the nearest 0.3 meters. Our apparatus was used to recompress and Water quality was monitored externally and decompress 14 cowcod. The fish were recompressed internally of the chamber. Externally water quality to pressures equivalent to 84.7 m ±2.2 m in depth. was monitored downstream of each chamber using an The average time taken to place the fish in the Oxyguard Handy Gamma probe. The probe was chamber and seal the lid on the chamber was 8.0 factory fit with temperature (±0.1°C) and dissolved minutes ±1.4 minutes and the average time taken to oxygen (±0.1%). Measurements were recorded at recompress the fish to the desired pressure was 1.8 hourly intervals while on the boat and every few minutes ±0.9 minutes. Recompression rates ranged hours in the laboratory during the daytime. from 20.5 to 164.3 m/min with an average of 63.6 cm Measurements and observations were also recorded ±37.6 m. at night between staged decompression plateaus. For Following recompression, the cowcod were continuous in-chamber measurements an Onset allowed to stabilize for 20 to 25.5 hours. After that Tidbit temperature probe was deployed to record period fish were decompressed at a rate of 1.4 m/min temperature every 30 seconds to the nearest 0.01°C. to sequential target stabilization depths (plateaus). No other water quality parameters were measured for The stabilization depths typically were 75.8 m ±0.6 the chambers. m, 60.4 m ±0.5 m, 47.0 m ±0.2 m, 31.5 m ±0.5 m, 18.0 m ±0.2 m, 11.1 m ±0.4 m, 4.1 m ±0.3 m, and 3.6 Decompression sequence. Fish were stabilized at m ±1.0 m. Stabilization times at those depths ranged depth for 20 to 25.5 hours prior to beginning their from 5.4 to 11.0 hours depending on scheduling and decompression sequence. Four to six hours into the initial stabilization depth. Total recompression initial stabilization period the chambers were isolated decompression treatments lasted from 73.4 to 150.4 (valves closed and disconnected from the water hours and produced healthy viable specimens (Figure source on the boat) and transported back to the 5). laboratory. Transportation times took no longer than 25 minutes, at which point water flow was reestablished to the chambers from the laboratory supply system. After the stabilization period, fish were decompressed manually in stages typical of those performed by scuba divers practicing a safety stop. This routine allowed for a relatively rapid change in pressure with an extended period of time to decompress at the new pressure. This extended stabilization period allowed fish to equalize internal gas concentrations with those of the environment, reducing the possibility of bubble formation in the circulatory system. Ascent rates used to calculate minimum time required to “off gas” varied from 1.8 to 0.9 m/min Figure 5. Decompression profile for one of 14 cowcod depending on depth. Ascent rates were: 1.8 m/min successfully collected by HSWRI. Pressure readings converted to actual or simulated depths with water below 61.0 m, 1.5 m/min between 30.5 and 61.0 m, temperature also shown. 1.2 m/min between 15.2 and 30.5 m, and 0.9 m/min between 0 and 15.2 m. Water temperature. Variation in water temperature Termination of decompression. At the end of each in the chamber was dependent on the operation of decompression sequence, the chamber was set chillers both on the boat and in the laboratory. On vertically (blind flange up) and the blind flange was the boat the initial temperature in the chamber was removed. The fish was then sluiced into a custom- warmer than desired because chilled seawater was built, vinyl sling and placed into a 1.5 m diameter not circulated through the chambers while waiting for pool. The chambers were cleaned with freshwater fish to be captured. During the recompression and scrubbed as needed between uses. Maintenance sequence chilled water was circulated and the of the system was also conducted at this time. temperature inside the chamber quickly decreased from a maximum of 18°C to the target temperature of

60 Smiley approximately 12°C. This usually occurred in the References first 30 minutes of operation and thereafter the Beyer, D. L., B. G. D’Aoust, and L.S. Smith. 1976. temperature stayed relatively constant. Decompression-induced bubble formation in salmonids: Comparison to gas bubble disease. Undersea Biomed. Res. 3(4):321–338. Conclusion The hyperbaric chamber was used successfully to Feathers, M. G., and A. E. Knable. 1983. Effects of recompress, transport, and decompress a species depressurization upon largemouth bass. N Am. J. Fish. never before maintained in captivity. The apparatus Manag. 3:86–90. is portable and capable of stable operation under a variety of field and laboratory conditions. The Gitschlag, G. R., and M. L. Renaud. 1994. Field system is also relatively inexpensive to build, experiments on survival rates of caged and released red operate, and maintain. Further modifications to the snapper. N. Am. J. Fish. Manag. 14:131–136. hatch design are being evaluated to expedite the process of inserting the fish and initiating the Gotshall, D. W. 1964. Increasing tagged rockfish (genus Sebastodes) survival by deflating the swim bladder. Calif. recompression sequence. This modification may also Fish Game 50:253–260. facilitate future attempts to use the chamber under water, so that fish do not need to ascend all the way Jannasch, H. W., C. O. Wiren, and C. L. Winget. 1973. A to the surface before being placed in the chamber. In bacteriological pressure-retaining deep-sea sampler and the future we plan to collect other species for culture vessel. Deep-Sea Res. 20:661–664. breeding purposes. We also plan to use the chambers in the laboratory to conduct studies to determine Keniry, M. J., W. A. Brofka, W. H. Horns, and J. E. species and size-specific differences in susceptibility Marsden. 1996. Effects of decompression and puncturing to barotrauma. the gas bladder on survival of tagged yellow perch. N. Am. J. Fish. Manag. 16:201–206.

Koyama, S., T. Miwa, M. Horii, Y. Ishikawa, K. Horikoshi, and M. Aizawa. 2002. Pressure-stat aquarium system designed for capturing and maintaining deep-sea organisms. Deep-Sea Res. 49:2095–2102.

Lee, D. P. 1992. Gas bladder deflation of depressurized largemouth bass. N. Am. J. Fish. Manag. 12:662–664.

Macdonald, A. G, and I. Gilchrist. 1978. Further studies on the pressure tolerance of deep-sea crustacean, with observations using a new high-pressure trap. Mar. Biol. 45:9–21.

Rogers, S. G., H. T. Langston, and T. E. Targett. 1986. Anatomical trauma to sponge-coral reef fishes captured by trawling and angling. Fish. Bull. 84:697–704.

Wilson, R. R. Jr., and K. L. Smith Jr. 1985. Live capture, maintenance, and partial decompression of a deep-sea grenadier fish (Coryphaenoides acrolepis) in a hyperbaric trap-aquarium. Deep-Sea Res. 32:1571–1582.

Yayanos. A. A. 1978. Recovery and maintenance of live amphipods at a pressure of 580 bars from an ocean depth of 5700 meters. Science 200:1056–1059.

Smiley 61

A Preliminary Report on the Use of Porphyra Protoplast as a Food Substitute for Culturing Aquatic Animals

TakaoYoshimatsu National Research Institute of Aquaculture Fisheries Research Agency Mie 516-0193, Japan [email protected]

Alok Kalla and Toshiyoshi Araki Mie University Graduate School of Bioresources Mie 514-8507, Japan

Dong-Ming Zhang Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences Fukuoka, 812-8581, Japan

Shuichi Sakamoto Oriental Yeast Industry Co. Ltd. Tokyo 174-8505, Japan

Keywords: Porphyra, protoplast, feed, red sea bream, Manila clams

Abstract Protoplasts are plant cells that have had their cell wall removed using either mechanical or enzymatic means. As protoplasts have no cell walls they are easily digestible when ingested by animals as food components. In the present experiment we investigated the availability of Porphyra protoplasts (PP) as a food supplement or live food substitute for culturing red sea bream and Manila clams in the laboratory. With respect to red sea bream, a PP supplemented diet improved the feed utilization efficiency and enhanced fish growth, although no remarkable effect was noted on carcass composition in either treatment group. Survival rate was improved by incorporating 5% PP in the diet. In Manila clams, high survival was obtained in both PP and diatom fed group. There appeared to be no difference in carcass composition between two adult shellfish groups fed on either PP or a natural diet. According to the experimental results obtained in these trials, Porphyra protoplasts proved to be a good food substitute candidate for culturing aquatic animals like finfish and bivalve spat.

Introduction Purple laver, Porphyra spp., is known to be one producing protoplasts from macroalgae using purified of the most nutritious edible macroalgae (red algae) polysaccharases isolated from various bacteria were and its thin sheet-like processed products are well developed (Araki et al. 1987, 1998, 2000; Aoki et al. known as a wrapping material for a famous Japanese 1990). As a result, the practical mass production of dish, sushi-rolls. Porphyra contains very high levels PP became a reality. of various kinds of minerals, and vitamins such as Protoplasts are plant cells that have had their cell vitamins A, B complex, C, and E. Laver is also a walls completely removed using either mechanical or good source of digestible protein (Resource Council enzymatic means. Spheroplasts are generally called 2000). In addition, it is notable that Porphyra something other than protoplasts because the cell contains a high amount of taurine, an important wall tends not to be destroyed completely by amino acid for larval fish (Kim et al. 2005). enzymatic treatment and some parts of the outer cell Recently, efficient biotechnological techniques for wall still remain intact. As protoplasts have no cell Yoshimatsu 63 walls, they are easily digestible when ingested by fish were weighed individually after being animals as a food ingredient. In the present anesthetized with 2-phenoxyethanol (500 mg/L). experiment we investigated the availability of Porphyra protoplasts (PP) as a live food substitute Table 1. Ingredient content and proximate analyses of the for culturing two types of aquatic animals: finfish and experimental diets for red sea bream. bivalve molluscs.

Materials and Methods The PP were prepared in the laboratory as follows. As the cell wall of this alga is composed of three kinds of polysaccharides (β-1, 4-mannan, β-1, 3-xylan, and porphyran), three enzymes (β-1, 4- mannanase; β-1, 3-xylanase; and agarase) were produced from various types of bacteria which have been isolated from marine environments (Araki et al.

1987, 1998, 2000; Aoki et al. 1990). Suitable conditions for preparing a large amount of PP in the laboratory were determined before the onset of rearing experiments. Included were determinations At the end of the feeding trials, in addition to the of the proper pH of the reaction mixture, body weight and length measurements, hepato- and concentration of each enzyme, and time and viscero-somatic indices were calculated from the temperature of the reaction process, among others. weights of individual bodies, livers, and viscera. Since it is particularly difficult to distinguish and Proximate analyses of liver and dorsal muscle were separate protoplasts from spheroplasts in a whole carried out from pooled samples of each group reaction mixture, we use only the term protoplasts obtained after termination of the experiment. The here when referring to combined enzymatic-treated biological parameters used for evaluating rearing product from Porphyra. After obtaining the results were defined and calculated as follows: protoplasts, they were subjected to freeze drying so Condition factor = body wet weight (g) x the nutrient qualities were retained. Freeze-dried PP 1000/body length3 (cm). were ground and smashed into powder form Hepatosomatic index (HSI) = liver wet weight manually using a mortar pestle. The particle size of (g) x 100/body wet weight (g). PP after grinding was varied from several µm to Viscerosomatic index (VSI) = whole visceral wet several tens of µm depending on the level of weight (g) x 100/body wet weight (g). enzymatic reaction that had occurred. Daily growth rate = (ln Wf-ln Wi) x 100/rearing One hundred liter polycarbonate aquaria were period (days), where Wf and Wi are final and stocked with 13 fish each (initial average body initial mean wet body weights (g), weight = 15.4 g ±0.2 g) that were fed the respectively. experimental diets for 6 weeks. Each aquarium was Feed efficiency = body wet weight gain x aerated (400–600 mL/min) and supplied with flow 100/dry weight of feed fed (g). through temperature-controlled seawater (32–34 ppt, Protein retention rate = dorsal muscle protein 25°C) at 8 L/min under the artificial photoperiod of gain (g) x 100/protein fed (g). 12L:12D. Two kinds of semipurified diets (Table 1) Protein efficiency ratio = body wet weight gain with and without supplementation of 5% PP were (g)/protein fed (g). formulated using brown fish meal as a major protein source (crude protein level after formulation: 48.7– The trials with bivalves were conducted as 51.0%. Every morning the diets were processed into follows. Manila clam (Ruditapes philippinarum) spat moist material by adding 60% w/w of freshwater. were obtained from a hatchery. Spat (6-months-old, They were fed ad libitum three times a day at 1000, mean shell length 5.34 mm) were randomly 1400, and 1800 hours by hand. The daily feeding distributed in small stainless steel mesh cages (10 cm ration was about 6% of the mean wet body weight diameter), with 20 individuals each in six replicates estimated from the growth curve obtained during receiving each dietary treatment and reared in a biweekly measurement when all the experimental flowthrough system of temperature controlled seawater (32–34 ppt, 20°C) for 6 weeks. Two diets

64 Yoshimatsu (PP and condensed natural diatoms [Chaetoceros PP and control fish showed reduced moisture content gracilis] Tasaki Shinju Co. Ltd., Japan) were fed ad and increased crude protein and lipid content. libitum to the clams four times a day (0800, 1200, However, there were no significant differences in 1600, and 2000 hours) by stopping the water flow proximate analyses data between them (p > 0.05). until clams complete consuming the food. Adult clams (mean shell length 35.3 mm) were reared under Table 3. Effect of Porphyra protoplast supplementation on the same conditions as those used with spat and for the growth and feed utilization by red sea bream. the same period. Before feeding, the PP was mixed with sea water using an electric mixer and offered to the clams in liquid form. The dietary performance was evaluated by growth of the shells, body composition and survival.

Results Proximate compositions of dried Porphyra and the PP are shown in Table 2. After enzymatic processing of Porphyra the protein and lipid contents increased while crude ash content decreased due mainly to the digestion of the cell wall polysaccharides.

Table 2. Proximate composition of Porphyra protoplasts. Table 4. Proximate compositions of the dorsal muscle of red sea bream fed on Porphyra protoplast. Figure 1. Increase in shell length of spat fed PP as compared with diatoms over a 6 week experimental period.

The rearing results of red sea bream fed on test diets with (PP) or without PP supplementation (control) A review of preliminary trial data on length are summarized in Table 3. After 6 weeks of feeding increase indicated that clams fed on natural diatoms the fish reached 65.9 g ±3.5 g in the PP group, but grew faster than the clams fed the PP diet (Figure 1, p only 57.0 g ±2.1 g in the control group. There was < 0.01). Nevertheless, over-all mortality was low more than a 60% difference in the percentage of (97.5–99.2%) and independent of the experimental average weight gained between the PP and control treatments. Also, no significant variation was groups (p < 0.01). As a result, a significantly better observed in proximate carcass composition after the (p < 0.01) daily growth rate (3.47% ±0.16%) was feeding trial for adult clams (Table 5). After 6 weeks obtained in the PP group compared with the control rearing, both PP and natural diatom-fed groups group (3.11% ±0.11%). In addition, survival was showed reduced moisture content and increased better in PP group (94.9–100%). As for feed crude protein and lipid contents; the same result as utilization, the PP group showed better results in feed obtained in red sea bream. efficiency, protein retention rate, and protein efficiency than the control group, although the two test diets had no major differences in their proximate compositions (Table 2). There was no significant difference in condition factor between the PP and control groups. Nevertheless, higher values were obtained in both HSI and VSI for the PP group. In Table 4, proximate compositions of the dorsal muscle sampled from test fish after the termination of the feeding trial are shown. After 6 weeks feeding, both

Yoshimatsu 65 system or activating fat accumulation/mobilization as well as improving absorption, assimilation, and retention (Mustafa et al. 1995). Nevertheless, reports of the supplemental effect of microalgal protoplasts have apparently not been published to date. Enhanced feed efficiency of red sea bream obtained in the present experiment, when fed with diet supplemented with 5% PP, may also be attributed to the probable presence of a food attractants, vitamins, minerals, essential amino acids, and taurine. Also, physiological changes induced through dietary PP supplementation might have affected the feeding behavior and efficiency of the test fish. At the moment, we do not know the mechanism by which Figure 1. Increase in shell length of spat fed PP as compared PP supplementation improved the dietary value of the with diatoms over a six week experimental period. feed for red sea bream in this feeding trial. Further study is needed to clarify this point. As for the results obtained in the clam Table 5. Proximate composition of Manilla clam fed on Porphyra protoplast and Chaetoceros gracilis. experiment, PP proved to be a good food source for culturing not only grown shellfish but also spat. Under the experimental conditions employed, growth in PP group of spat was inferior to that of spat fed diatoms (Figure 1). Nevertheless, final survival was high and almost same in the PP-fed group as in natural food group. As there appeared to be no difference in carcass composition between the two groups, it should be possible in the future to improve the dietary effect by manipulating feeding techniques and ingredients added to PP diet to enhance its quality. According to the experimental results obtained in Discussion these culture trials, the protoplasts of Porphyra In red sea bream, a PP supplemented diet proved to be a good food substitute for culturing improved feed utilization efficiency and enhanced aquatic animals like finfish and bivalve spat. We fish growth (Table 3), although no notable effects should continue investigating the applicability of PP were obtained with respect to carcass composition of and determine what kind of nutrients must be the fish used in the two treatments (Table 4). Dietary supplemented to enhance the dietary value to meet protein utilization was improved significantly by the nutritional requirements of these aquatic animals supplementing PP as shown in Table 3. As a feed and develop the feeding techniques that might ingredient, protein is the most important dietary enhance the performance of the PP. element and its efficient utilization must be thought of as the first priority when formulating a fish food. Acknowledgment The experimental results obtained in this feeding trial This study was supported by a grant program of show that PP has great potential as a food supplement the Agriculture, Forestry, and Fisheries Research from the viewpoint of efficient utilization of dietary Council in Japan (Research project for utilizing protein. Survival rate was also improved by the advanced technologies in agriculture, forestry, and supplementation of 5% PP to the diet. fisheries. No.1681, 2004-2006, Studies on the Nakagawa (2005) summarized information effective utilization of low-grade Porphyra by indicating that small amounts of various kinds of enzymatic means). macroalgal supplementation to the diet improves fish growth and feed efficiency, mainly due to an improvement in lipid metabolism. Algal supplementation to fish feed was previously reported to be effective either by enhancing the immune 66 Yoshimatsu References Aoki, T., T. Araki, and M. Kitamikado. 1990. Kim, S.K., T. Takeuchi, A. Akimoto, H. Furuita, T. Purification and characterization of a novel beta-agarase Yamamoto, M. Yokoyama, and Y. Murata. 2005. Effect from Vibrio sp. AP-2. Eur. J. Biochem. 187:461–465. of taurine supplemented practical diet on growth performance and taurine contents in whole body and Araki, T., T. Aoki, and M. Kitamikado. 1987. Preparation tissues of juvenile Japanese flounder Paralichthys and regeneration of protoplasts from wild-type of olivaceus. Fish. Sci. 71:627–632. Porphyra yezoensis and green variant of P. tenera. Nippon Suisan Gakk. 53:1623–1627. Mustafa, M. G., S. Wakamatsu, T. Takeda, T. Umino, and H. Nakagawa. 1995. Effects of algae meal as feed Araki, T., Z. Lu, and T. Morishita. 1998. Optimization of additive on growth, feed efficiency, and body composition parameters for isolation of protoplasts from Gracilaria in red sea bream. Fish. Sci. 61:25–28. verrucosa (Rhodophyta). J. Mar. Biotechnol. 6:193–197. Nakagawa, H. 2005. Effective utilization of macroalgal Araki, T., S. Hashikawa, and T. Morishita. 2000. waste as feed ingredients for aquaculture. In M. Sakaguchi Cloning, sequencing, and expression in Escherichia coli of and T. Hirata (eds.), Advanced effective utilization of fish the new gene encoding β-1, 3-xylanase from a marine and fisheries products, p. 292–300. NTS Press, Tokyo, bacterium, Vibrio sp. strain XY-214. Appl. Environ. Japan. Microbiol. 66:1741–1743. Resource Council. 2000. Algae. In Standard tables of Halver, J. E. 1957. Nutrition of salmonoid fishes–III. food composition in Japan (Fifth revised edition), p. 152- Water-soluble vitamin requirements of Chinook salmon. J. 159. Science and Technology Agency, Tokyo, Japan. Nutr. 62:225–243.

Yoshimatsu 67

Aquaculture and Stock Enhancement Technologies Based on Recently Discovered Calcium-sensing Receptors in Finfish

Steve Jury, H. William Harris, Timothy Linley, David Russell, Marlies Betka, and Susan Anderson MariCal Inc. Portland, Maine USA (www.marical.biz) [email protected]

Shinji Harakawa and Akikuni Hara Hakuju Institute for Health Science, Tokyo, Japan (www.hakuju.co.jp)

Keywords: calcium sensing receptor, salinity, enhancement, salmon, cobia, aquaculture

Extended Abstract Recent discoveries that calcium-sensing receptors (CaRs) act as salinity sensors and are involved in sensory reception in finfish provides a basis for the development of multiple technologies designed to improve aquaculture and stock enhancement efforts in the U.S. and Japan. The combination of molecular cloning, cell biology, and physiological experiments show that CaRs play key roles in finfish osmoregulation and physiological adaptation to varying salinity environments. Important CaR-mediated roles in finfish appear to involve not only osmoregulatory acclimation via gill, renal and intestinal tissues, but also changes in endocrine as well as olfactory responses. Taken together, these data provide support for CaRs as regulators of the multiple physiological and behavioral changes occurring during smoltification in salmonid species. Based on knowledge of the ionic pharmacology of CaRs, juvenile salmonids can be pre-adapted to seawater while they remain in freshwater. Alternatively, marine fish species normally restricted to seawater can be maintained under near freshwater conditions. In this regard, the SuperSmolt and SeaReady processes are currently being licensed to salmon aquaculture and stock enhancement producers. Both of these technologies are applied to fish reared in tanks where mineral salts are added to the rearing water and fish are fed a diet containing elevated NaCl and a naturally occurring CaR-reactive amino acid for an interval of 3–6 weeks. Thus the resulting biological changes are induced by manipulation of natural processes and do not involve genetic modifications. These changes are monitored using enzymatic (e.g., gill Na+K+ATPase) and physiological (e.g., seawater challenge, plasma chloride level) tests to determine development of seawater tolerance. In farmed Atlantic salmon (Salmo salar), the SuperSmolt process reduces the deleterious effects of incomplete smoltification after the transfer of smolts to seawater. This improves both the growth and feed conversion ratios while reducing mortalities during growout in seawater. In Chinook, coho, and sockeye salmon (Oncorhynchus tshawytscha, O. kisutch, O. nerka), the SeaReady process improves the osmoregulatory and growth performance in the early seawater rearing environment and preliminary data suggest that this may increase the number and performance of adult returns. The SuperSmolt process has been recently tested and endorsed by EWOS, a major aquaculture feed producer. By contrast, CaR modulation in marine fish such as cobia (Rachycentron canadum) enables their production under very low salinity conditions, providing inland growers or those utilizing recirculation technologies the option to produce high value marine species. Research by MariCal and Hakuju has also focused on the roles of CaRs in sensory biology, including salinity reception, olfaction, and the response of fish to electric fields via lateral line or specific electroreceptor tissues. Since a gradient of charged ions (Na+, Cl-, Mg2+ or Ca2+) is likely involved in salinity as well as electric field detection, CaRs in specific tissues may provide a molecular means to “sense” and integrate changes in both the ionic and electric field environments that fish may encounter. While much of this research is ongoing, we conclude that this CaR-based approach has considerable potential to improve aquaculture and stock enhancement technologies in the U.S. and Japan.

Jury 69 Background. CaRs are part of a family of G-protein coupled receptors that are closely related to metabotropic glutamate and pheremone receptors and are highly conserved between all species tested (Breitwieser et al. 2004, Ingleton et al. 1999, Figure 1). CaR proteins were first identified in human parathyroid glands and their role in calcium homeostasis has been firmly established (Brown and MacLeod 2001, Breitwieser et al.2004, Chang and Shoback 2004). CaRs are responsive to physiologically and environmentally relevant changes in extracellular divalent cations such as Ca2+ and Mg2+ (Breitwieser et al. 2004), but are also modulated by other extracellular conditions including NaCl (Quinn et al. 1998, Loretz et al. 2004), specific amino acids (Conigrave et al. 2004), several heavy 2+ 3+ metals; e.g., Pb and Al (Handlogten et al. 2000, Quinn et al. 2004), and pH (Doroszewicz et al. 2005, Quinn et al. 2004). CaRs have been found in Figure 1. Calcium-sensing receptors (CaR) are integrators of extracellular and environmental signals leading to various multiple organs associated with osmoregulation and intracellular responses. salinity detection in several fish species including Atlantic salmon (Salmo salar)(Nearing et al. 2002, Nearing et al. submitted), sea bream (Sparus aurata) this report we describe applications developed using (Flanagan et al. 2002), and tilapia (Oreochromis this approach as well as an introduction to some mossambicus)(Loretz et al. 2004). We have also research to develop additional technologies. These characterized a full-length CaR cDNA from dogfish applications are: 1) SeaReady, 2) growing marine (Squalus acanthias) kidney (SKCaR) that is localized fish in near freshwater, and 3) the role of CaR in in kidney, olfactory lamellae, epithelial cells and sensory biology. vessels in the gastrointestinal tract, gills, and rectal gland (Nearing et al. 2002, Hentschel et al. 2003). SeaReady. Anadromous species, including Several studies have shown CaR in other fish species salmonids, are able to survive in either freshwater or without molecular cloning of their full-length seawater due to osmoregulatory adjustments in transcripts or proteins. These species include the tissues including gill, skin, kidney, and flounders, Platichthys flesus (Ingleton et al. 1999) gastrointestinal tract. These are the same tissues and Pleuronectes americanus (Nearing et al. 2002), where CaRs are localized (Nearing et al. submitted) and goldfish, Carassius auratus (Hubbard et al. and may be differentially expressed during seawater 2002). 2+ acclimation. The process of smoltification in The potential role of CaRs as regulators of Ca salmonids raised under hatchery production homeostasis in fish has been emphasized (Hubbard et conditions prepares salmon for the remodeling of al. 2000, 2002, Ingleton et al. 1999, Radman et al. their tissues after seawater transfer. These 2002, Fellner and Parker 2002, Loretz et al. 2004, physiological changes must be accomplished within a Nearing et al. submitted), but many of these studies limited interval of time and be matched to the have also suggested the involvement of CaRs in movement of fish to higher salinity environments. salinity sensing in fish (Nearing et al. 2002, Hubbard These changes may occur naturally during a normal et al. 2002, Loretz et al. 2004, Nearing et al. smolt timing window that is dependent on the species submitted). concerned. The complex changes that occur during Thus CaRs are present and functional in various smolting determine how ready salmonids are for a tissues of multiple teleost species, and we propose seawater existence. Smoltification has traditionally that they are important in the integration of several been manipulated under hatchery rearing conditions physiological and behavioral processes of concern to with varying degrees of success by changes in the aquaculture and stock enhancement industries. photoperiod, temperature, and feeding (McCormick We have used the approach of studying receptor et al. 1998). While the factors that initiate and biology to address problems of concern in hatchery control smoltification continue to be the subject of production of anadromous and marine species. In 70 Jury many studies, they are not entirely understood. The growth during seawater rearing. This process has variability in the effects of different methods, as well been successfully tested on multiple species (Atlantic as logistic problems associated with hatchery rearing salmon, Chinook, coho, and sockeye salmon) in often leads to a gradient of incomplete smoltification multiple locations on over 3 million smolts. We and subsequent osmotically induced lethal and believe that there may be additional effects of this sublethal effects upon transfer of fish to seawater. preadaptation process, including priming for These effects can include significant elevations in imprinting, manipulating residualization, decreased plasma ionic content resulting in the dysfunction of estuarine residency time (and thus decreased various organ systems, impaired ion homeostasis, predation in early marine life history), as well as abnormal swimming behavior, morphological improved ocean survival and subsequest improved changes, impaired feeding behavior, and often returns of released fish (Linely 2000). However, increased mortality. these effects remain to be quantified. Based on our understanding of the distribution and function of calcium-sensing receptors relative to Growing marine fish in near freshwater. osmoregulation, as well as the logistic and Acclimating marine species to grow in freshwater or socioeconomic constraints of hatchery operations, near freshwater conditions offers multiple benefits Marical has developed the SeaReady (U.S. patent for hatchery production. For certain applications, #6,748,900) and SuperSmolt (U.S. patent freshwater recirculating systems are more feasible on #6,463,883) processes as methods to enhance a commercial scale to raise marine species, then smoltification in salmonid fishes while they are still transfer them to marine sites for growout. This in the hatchery prior to release into rivers or transfer would allow for raising fish to a larger size under to seawater for growout. This process consists of controlled hatchery conditions, potentially at a lower manipulating the ionic environment (e.g., Ca2+ and cost. The reasons for using freshwater systems Mg2+) as well as application of feed addititives (NaCl include: 1) coastal property is very expensive for and CaR reactive amino acids) over a period of 4–6 shore based tank farms and political/environmental weeks (Figure 2). pressures are restrictive along the coast in many areas, 2) environmental regulations limit the amount of saline water discharge and sludge disposal from recirculating facilities, 3) higher value marine species may be grown in systems currently culturing lower value species, and 4) freshwater culture of marine species may mitigate some disease issues. We have patented (U.S. patent # 6,463,882) methods to grow marine species in near freshwater by manipulating the ionic composition of the rearing environment and adding CaR compounds to the feed to induce compensatory changes in the plasma. The fish are subjected to a gradual or step-wise decrease

in salinity for a period of time prior to transfer to Figure 2. SeaReady process overview. freshwater, while being fed a modified diet. The fish are then maintained in a near freshwater low salinity environment with an optional long day photoperiod SeaReady serves to preadapt salmon to seawater for a sufficient period of time to increase or maintain and allows for improved performance upon transfer expression or sensitivity of calcium-sensing to seawater. In terms of stock enhancement, we receptors. propose that this improved early seawater MariCal has been conducting feasibility studies performance will lead to enhanced ocean survival with cobia (Rachycentron canadum) and has (Linley 2000) and subsequent improved returns. determined that cobia grow well in salinities as low Benefits include the ability to transfer fish to as 2 ppt and can be successfully transferred from seawater at smaller sizes, transferring of smolts these conditions back to 35 ppt seawater. MariCal outside of their traditional species specific smolt plans to grow cobia to market size in future large timing windows (e.g., fall entry), reduced feeding scale trials. inhibition upon seawater transfer, and improved

Jury 71 Role of CaRs in sensory biology. CaRs are References expressed in most osmoregulatory tissues in salmon, Breitweiser, G. E., S. U. Medlich, and M. Zhang. 2004. but they are also found throughout the peripheral and Calcium sensing receptors as integrators of multiple central nervous systems (Nearing et al. 2002, Nearing metabolic signals. Cell Calcium 35:209–216. et al. submitted). In particular CaRs have been found in the olfactory system (olfactory epithelia, olfactory Brown, E. M., and R.J. MacLeod. 2001. Extracellular bulb, olfactory nerve) as well as in electroreceptive calcium sensing and extracellular calcium signaling. Physiol. Rev. 81:239–297. organs (e.g., electric organs, ampullae of Lorenzini) of teleosts and elasmobranchs. Based on extra- Chang, W., and D. Shoback. 2004. Extracellular Ca2+- cellular recordings of the olfactory nerve, we have sensing receptors-an overview. Cell Calcium 35:183–196. determined that Atlantic salmon are responsive to calcium, magnesium, gadolinium, and strontium at Conigrave, A. D., H. Mun, L. Delbridge, S. J. Quinn, M. concentrations that are consistent with CaR activation Wilkinson, and E. M. Brown. 2004. L-amino acids or modulation (Nearing et al. 2002, Nearing et al. regulate parathyroid hormone secretion. J. Biol. Chem. submitted). On this basis, we propose that CaRs play 279:38151–38159. a role in multiple osmoregulatory, and sensory functions in salmon which experience large changes Doroszewicz, J., P. Waldegger, N. Jeck, H. Seyberth, and S. Waldegger. 2005. pH dependence of extracellular in the concentration and composition of the ionic calcium sensing receptor activity determined by a novel environments encountered during their normal life technique. Kidney Int. 67:187–192. history. This has many implications for stock enhancement of salmonids including the role of CaRs Fellner, S. K., and L. Parker. 2002. A Ca(2+)-sensing in olfactory mediated behaviors such as imprinting, receptor modulates shark rectal gland function. J. Exp. homing, mate selection, and predator avoidance. Our Biol. 205:1889–1897. research programs have focused on the function of CaR in marine and aquatic organisms. Using Flanagan, J. A., L. A. Bendell, P. M. Guerreiro, M. S. information from these studies, we plan to continue Clark, D. M. Power, A. V. M. Canario, B. L. Brown, and developing applications useful to the stock P. M. Ingleton. 2002. Cloning of the cDNA for the putative calcium-sensing receptor and its tissue enhancement and aquaculture industries. distribution in sea bream (Sparus aurata). Gen. Comp. Endocr. 127:117–127. Conclusions. The identification of CaRs in multiple tissues of many species of finfish is an important Handlogten, M. E., C. Huang, N. Shiraishi, H. Awata, and discovery opening an area of research on the R.T. Miller. 2000. Extracellular Ca2+-sensing receptor is a integration of environmental and extracellular promiscuous divalent cation sensor that responds to lead. signalling of cations. Exposure of finfish to variable Am. J. Physiol.-Renal. 279:F1083–1091. ionic environments under naturally occurring conditions (e.g., the typical life history of Hentschel, H., J. Nearing, H. W. Harris, M. Betka, M. anadromous species) or under hatchery conditions Baum, S. C. Hebert, and M. Elger. 2003. Localization of Mg2+-sensing shark kidney calcium receptor SKCaR in (e.g., SeaReady) influences CaR signaling in specific kidney of dogfish, Squalus acanthias. Am. J. Physiol.- tissues (e.g., gill, olfactory epithelia, electric organ). Renal. 285:F430–439. CaRs are likely to be involved in many physiological functions in the salmonid life cycle, and using an Hubbard, P. C., E. N. Barata, and A. V. Canario. 2000. evolving understanding of the role of these receptors, Olfactory sensitivity to changes in environmental [Ca(2+)] we have developed technologies beneficial to the in the marine teleost Sparus aurata. J. Exp. Biol. stock enhancement and aquaculture industries in the 203:3821–3829. U.S. and Japan. Hubbard, P. C., P. M. Ingleton, L. A. Bendell, E. N. Barata, and A. V. Canario. 2002. Olfactory sensitivity to changes in environmental [Ca(2+)] in the freshwater teleost Carassius auratus: An olfactory role for the Ca(2+)-sensing receptor? J. Exp. Biol. 205:2755–2764.

72 Jury Ingleton, P. M., P. C. Hubbard, J. A. Danks, G. Elgar, R. Nearing, J., M. Betka, S. Jury and H.W. Harris. A. Sandford, and R. J. Balment. 1999. The calcium- Submitted. Atlantic salmon (Salmo salar) express sensing receptor in fishes. In J. Danks, C. Dacke, G. Flik, calcium-sensing receptors (CaRs) in various tissues. J. and C. Gay (eds.), Calcium metabolism: Comparative Exp. Biol. endocrinology, p. 45–48. BioScientifica Ltd., Bristol, UK. Quinn, S. J., O. Kifor, S. Trivedi, R. Diaz, P. Vassilev, and Linley, T. 2000. Influence of short-term estuarine rearing E. Brown. 1998. Sodium and ionic strength sensing by on the ocean survival and size-at-return of coho salmon in the calcium receptor. J. Biol. Chem. 273:19579–19586. southeast Alaska. N. Am. J. Aquacult. 63:306–311. Quinn, S. J., M. Bai, and E. M. Brown. 2004. pH sensing Loretz, C. A, C. Pollina, S. Hyodo, Y. Takei, W. Chang, by the calcium-sensing recptor. J. Biol. Chem. and D. Shoback. 2004. cDNA cloning and functional 279:37241–37249. expression of a Ca2+-sensing receptor with truncated C- terminal tail from the Mozambique tilapia (Oreochromis Radman, D. P., C. McCudden, K. James, E. M. Nemeth, mossambicus). J. Biol. Chem. 279:53288–53297. and G. F. Wagner. 2002. Evidence for calcium-sensing receptor mediated stanniocalcin secretion in fish. Mol. McCormick, S. D., L. P. Hansen, T. P. Quinn and R. L. Cell. Endocrinol. 186:111–119. Saunders. 1998. Movement, migration and smolting of Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. Speca, D. J., D. M. Lin, P. W. Sorensen, E. Y. Isacoff, J. 55:77–92. Ngai, and A. H.Dittman. 1999. Functional identification of a goldfish odorant receptor. Neuron 23:487–498. Naito, T., Y. Saito, J. Yamamoto, Y. Nozaki, K. Tomura, M. Hazama, S. Nakanishi, and S. Brenner. 1998. Putative pheromone receptors related to the Ca2+-sensing receptor in Fugu. Proc. Natl. Acad. Sci. 95:5178–5181.

Nearing, J., M. Betka, S. Quinn, H. Hentschel, M. Elger, M. Baum, M. Bai, N. Chattopadyhay, E. M. Brown, S. C. Hebert, and H. W. Harris. 2002. Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish. Proc. Natl. Acad. Sci. 99:9231–9236.

Jury 73

Biological Studies on the Digestive Mechanism in Abalone

Kentaro Niwa and Hideaki Aono National Research Institute of Fisheries Science 6-31-1 Nagai, Yokosuka Kanagawa 238-0316, Japan. [email protected]

Tomoo Sawabe Laboratory of Microbiology Hokkaido University 3-1-1 Minato-cho, Hakodate Hokkaido 041-8611, Japan

Keywords: abalone, Vibrio halioticoli, alginate lyase

Abstract Abalone are one of the most important marine gastropods in Japan’s coastal fishery. Recent studies on abalone suggest that the gut microbes work together with polysaccharide degrading enzymes of the host to digest seaweeds in the stomach. Genes encoding alginate lyase have been cloned from the digestive diverticulum of Haliotis discus hannai and H. d. discus. The bacterium, Vibrio halioticoli, with alginate degrading activity has also been isolated from the digestive fluid of various abalone species. However, the process by which the bacterium colonizes gut of abalone is unknown. Therefore, our goal is to clarify when colonization occurs and to what extent the bacterium contributes to digestion of polysaccharides.

Introduction Abalone are economically important marine existence of alginate lyase in the digestive gastropods that bring moderate to high prices on the diverticulum of abalone (Tsujino and Saito 1962, world market (Oakes and Ponte 1996). In Japan, Nakada and Sweeny 1967, Ohnishi et al. 1985, there are six species of abalone, Haliotis discus Boyen et al. 1990). Uki and Watanabe (in Shepherd discus, H. discus hannai, H. madaka, H. gigantea, H. 1992) investigated the nutritional requirements for diversicolor diversicolor, and H. diversicolor the development of artificial feeds for abalone and aquatilis, that are targets of coastal fisheries and suggested that abalone can efficiently use alginic acid aquaculture. Abalone ingest mainly algae, with a as an energy source. It has been reported that preference for brown algae. Abalone are distributed alginate lyase detected from the digestive tract of along the coastal reefs in Japan, where many kinds of abalone preferentially degraded the polyM block kelp in the order Laminariales (brown algae) grow (Nakada and Sweeny 1967, Boyen et al. 1990). abundantly. Laminaria sp. is known to be the Alginate lyase specific for polyM was cloned from original source of alginate. Alginate accounts for the digestive diverticulum of H. discus hannai more than 30% of the dry weight of the thallus in (Shimizu et al. 2003). Recently, we succeeded in some algae. It is known that the cell walls of brown cloning of the equivalent gene from H. discus discus algae are formed by linkages of alginate and cellulose (in preparation). Furthermore, 68% of all alginolytic (Kloareg and Quatrano 1988). Alginate molecules bacteria isolated from H. discus hannai showed a consist of two homopolymeric structures: strong preference for the polyG block (Sawabe et al. polymannuronate (polyM) block and polyguluronate 1998). Therefore, it is reasonable to speculate that (polyG) block, and a heteropolymeric part, which is the polyM part of ingested alginate is degraded by the randomly connected to manuronate to guluronate digestive enzyme of the abalone, and the remaining (MG random block). These polysaccharides are polyG part is metabolized by these bacteria. difficult to degrade by vertebrate digestive enzymes. Sawabe et al. (1998) isolated a unique alginolytic However, some kinds of herbivorous arthropods, marine bacterium from the gut of H. discus hannai. nematodes and molluscs can degrade those The bacterium was identified as Vibrio halioticoli, polysaccharides. Many researchers have reported the which was recognized as a new species (Sawabe et Niwa 75 al. 1995). The bacterium produced at least six types References of alginate lyases, two of which were observed to be Boyen, C., B. Kloareg, M. Polne-Fuller, and A. Gibor. specific for polyM and four of which were specific 1990. Preparation of alginate lyase from marine mollusks for polyG. for protoplast isolation in brown algae. Phycologia V. halioticoli was detected from the gut of three 29:173–181. species of the Japanese abalone, H. discus discus, H. diversicolor aquatilis and H. diversicolor Breznak, J. A. 1982. Intestinal microbiota of termites and other xylophagous insects. Annu. Rev. Microbiol. 36:323– diversicolor, and the South African abalone, H. 343. midae, with a range of 40–65% among those microfloras (Sawabe et al. 2003). The bacterium was Kloareg, B., and R. S. Quatrano. 1988. Structure of the also found in the gut of a turban shell, Turbo cell walls of marine algae and ecophysiological functions cornutus, although the occupation rate was of the matrix polysaccharides. Oceanogr. Mar. Biol. Ann. significantly smaller than that found in Haliotis spp. Rev. 26:259–315. Furthermore, acetic acid and formic acid were detected as major fermentation products of alginate in Nakada, H. I., and P. C. Sweeny. 1967. Alginic acid the V. halioticoli strains. The acetate is absorbed degradation by eliminases from abalone hepatopancrease. from the gut tissue of abalone and metabolized as an J. Biol. Chem. 242:845–851. oxidative energy source (Breznak 1982). It is Oakes, F. R., and R. D. Ponte. 1996. The abalone market: suggested that the abundant populations of V. Opportunities for cultured abalone. Aquaculture 140:187- halioticoli in the gut of Haliotis abalone may have a 195. great role for converting alginate to acetic acid (Sawabe et al. 2003). Ohnishi, T., M. Suzuki, and R. Kikuchi. 1985. The The population of V. halioticoli in the gut of distribution of polysaccharide hydrolase activity in juvenile abalone increased when the diet was gastropods and bivalves. Bull. Jpn. Soc. Sci. Fish. 51:301– switched from diatoms to laminaria (Tanaka et al. 308. 2003). Although the finding seems to suggest that the main source of the bacterium is thalli of Sawabe, T., Y. Oda, Y. Shiomi, and Y. Ezura. 1995. Alginate degration by bacteria isolated from the gut of sea laminaria, V. halioticoli has never been isolated from urchins and Abalones. Microbial. Ecol. 30:193–202. that alga. The main route and timing of gut colonization are not yet known. Therefore, we plan Sawabe, T., I. Sugimura, M. Ohtsuka, K. Nakano, K. to clarify when V. halioticoli colonize the gut of the Tajima, Y. Ezura, and R. Christen. 1998. Vibrio abalone and to what extent the bacterium contributes halioticoli sp.nov., a non-motile alginolytic marine to digestion of polysaccharides. bacterium isolated from the gut of the abalone Haliotis discus hannai. Int. J. Syst. Bacteriol. 48:573–580.

Sawabe, T., N. Setoguchi, S. Inoue, R. Tanaka, M. Ootsubo, M. Yoshimizu, and Y. Ezura. 2003. Acetic acid production of Vibrio halioticoli from alginate: a possible role for establishment of abalone-V.halioticoli association. Aquaculture 219:671–679.

Shepherd, S. A. 1992. Abalone of the World, p. 504-517. Fishing News Books, Oxford, UK.

Shimizu, E., T. Ojima, and K. Nishita. 2003. cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai. Carbohydr. Res. 338:2841–2852.

Tanaka, R., I. Sugimura I., T. Sawabe, M. Yoshimizu, and Y.Ezura. 2003. Gut microflora of abalone Haliotis discus hannai in culture changes coincident with a change in diet. Fish. Sci. 68:227–229.

Tsujino, I., and T. Saito. 1962. Studies on alginase. Part II. A new unsaturated uronide isolated from alginase hydrolysate. Agr. Biol. Chem. Tokyo 26:115–118. 76 Niwa